Helicase-dependent amplification of RNA

ABSTRACT

Methods and a kit are provided for selectively and exponentially amplifying nucleic acids and include the use of a single strand helicase preparation or a thermostable helicase in the absence of a single strand binding protein and a DNA polymerase such that the amplification can be performed isothermally.

GOVERNMENTAL SUPPORT

This invention is partially supported by the National Institutes ofHealth under SBIR Grant #AI066487. The U.S. government has certainrights to the invention.

BACKGROUND

Amplification of nucleic acids is widely used in research, forensics,medicine and agriculture. One of the best-known amplification methods isthe polymerase chain reaction (PCR), which is a target amplificationmethod (See for example, U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159). A PCR reaction typically utilizes two oligonucleotideprimers, which are hybridized to the 5′ and 3′ borders of the targetsequence and a DNA polymerase which can extend the annealed primers byadding on deoxynucleoside-triphosphates (dNTPs) to generatedouble-stranded products. By raising and lowering the temperature of thereaction mixture, the two strands of the DNA product are separated andcan serve as templates for the next round of annealing and extension,and the process is repeated.

Although PCR has been widely used by researchers, it requiresthermo-cycling to separate the two DNA strands. Several isothermaltarget amplification methods have been developed in the past 10 years.One of them is known as Strand Displacement Amplification (SDA). SDAcombines the ability of a restriction endonuclease to nick theunmodified strand of its target DNA and the action of anexonuclease-deficient DNA polymerase to extend the 3′ end at the nickand displace the downstream DNA strand. The displaced strand serves as atemplate for an antisense reaction and vice versa, resulting inexponential amplification of the target DNA (See, for example, U.S. Pat.Nos. 5,455,166 and 5,470,723). In the originally-designed SDA, the DNAwas first cleaved by a restriction enzyme in order to generate anamplifiable target fragment with defined 5′ and 3′-ends but therequirement of a restriction enzyme cleavage site limited the choice oftarget DNA sequences (See for example, Walker et. al., Proc. Natl. Acad.Sci. USA 89:392-396 (1992)). This inconvenience has been circumvented bythe utilization of bumper primers which flank the region to be amplified(Walker et al. supra (1992)). SDA technology has been used mainly forclinical diagnosis of infectious diseases such as chlamydia andgonorrhea. One of the most attractive feature of SDA is its operation ata single temperature which circumvents the need for expensiveinstrumented thermal cycling. However, SDA is inefficient at amplifyinglong target sequences.

A second isothermal amplification system, Transcription-MediatedAmplification (TMA), utilizes the function of an RNA polymerase to makeRNA from a promoter engineered in the primer region, and a reversetranscriptase, to produce DNA from the RNA templates. This RNAamplification technology has been further improved by introducing athird enzymatic activity, RNase H, to remove the RNA from cDNA withoutthe heat-denaturing step. Thus the thermo-cycling step has beeneliminated, generating an isothermal amplification method namedSelf-Sustained Sequence Replication (3SR) (See, for example, Guatelli etal., Proc. Natl. Acad. Sci. USA 87:1874-1878 (1990)). However, thestarting material for TMA and 3SR is limited to RNA molecules.

A third isothermal target amplification method, Rolling CircleAmplification (RCA), generates multiple copies of a sequence for the usein in vitro DNA amplification adapted from in vivo rolling circle DNAreplication (See, for example, Fire and Xu, Proc. Natl. Acad. Sci. USA92:4641-4645 (1995); Lui, et al., J. Am. Chem. Soc. 118:1587-1594(1996); Lizardi, et al., Nature Genetics 19:225-232 (1998), U.S. Pat.Nos. 5,714,320 and 6,235,502). In this reaction, a DNA polymeraseextends a primer on a circular template generating tandemly linkedcopies of the complementary sequence of the template (See, for example,Kornberg and Baker, DNA Replication, W.H. Freeman and Company, New York(2^(nd) ed. (1992)). Recently, RCA has been further developed in atechnique, named Multiple Displacement Amplification (MDA), whichgenerates a highly uniform representation in whole genome amplification(See, for example, Dean et. al., Proc. Natl. Acad. Sci. USA 99:5261-5266(2002)).

Additional nucleic acid amplification methods include Ligase ChainReaction (LCR), which is a probe amplification technology (See, forexample, Barany, Proc. Natl. Acad. Sci. USA 88:189-193 (1991)); and U.S.Pat. No. 5,494,810), and branched DNA (bDNA) technology (Horn et al.,Nucleic Acids Res. 25:4842-4849 (1997)), which is a signal amplificationtechnology.

The amplification methods mentioned above all have their limitations.For example, PCR and LCR require a thermocycler with associatedinstrumentation. Except for PCR, none of the other target amplificationmethods are capable of amplifying DNA targets having sufficient lengthto be useful for cloning genes and analysis of virulence factors andantibiotic resistant genes. Although PCR is able to amplify a target upto 10-20 kb, high mutation rates may limit the use of PCR-amplifiedproducts (Cline et al., Nucleic Acids Res. 24, 3546-3551 (1996)). Thus,to minimize the problem, a high-fidelity amplification method for longtargets is needed. In addition, all present amplification methodsrequire prior heat denaturation and annealing steps to produce primedtemplates for DNA polymerases. This adds extra time to the amplificationprocess.

The potential uses for nucleic acid amplification techniques continuesto grow. For example, nucleic acid arrays frequently utilize largenumbers of amplification reactions. Detection of environmentalcontamination places demands on sensitivity and analytic power ofdiagnostic tests that include nucleic acid amplification procedures.Consequently, improvements in amplification methodology are desirable.

SUMMARY OF THE INVENTION

In an embodiment of the invention, a method is provided forexponentially and selectively amplifying a target nucleic acid thatincludes the steps of: providing single strand templates of the targetnucleic acid to be amplified; adding oligonucleotide primers forhybridizing to the templates; synthesizing an extension product of theoligonucleotide primers which are complementary to the templates, bymeans of a DNA polymerase to form a duplex; contacting the duplex with ahelicase preparation for unwinding the duplex; and repeating the abovesteps to exponentially and selectively amplify the target nucleic acid.

In additional embodiments of the invention, amplification may beisothermal and may be accomplished in the range of about 20° C.-75° C.,preferably at room temperature.

In additional embodiments of the invention, the target nucleic acid maybe either a single stranded nucleic acid, more particularly, a singlestranded DNA or a single stranded RNA, or a double stranded nucleicacid, more particularly a double stranded DNA. When the nucleic acid isdouble stranded, it may be denatured by heat or enzymatically to form asingle strand template for DNA polymerase dependent amplification. Inaddition, the target nucleic acid may have a size in the range of about50 bp to 100 kb.

In additional embodiments of the invention, the oligonucleotide primersused in the method of amplification are a pair of oligonucleotideprimers wherein one primer hybridizes to 5′-end and one primerhybridizes to 3′-end of the target nucleic acid to be selectivelyamplified. Under circumstances of multiplexing, multiple primer pairsmay be used to amplify multiple target nucleic acids in the samereaction mixture. In addition, the oligonucleotide primers may have alength and a GC content so that the melting temperature of theoligonucleotide primers is 10° C.-30° C. above the reaction temperatureof hybridization during amplification.

In additional embodiments of the invention, a DNA polymerase is selectedfrom a Klenow fragment of E. coli DNA polymerase I, T7 DNA polymerase(Sequenase) and Bst polymerase large fragment. Preferably, the DNApolymerase lacks 5′ to 3′ exonuclease activity and possesses stranddisplacement activity.

In additional embodiments of the invention, the helicase preparation mayinclude a single helicase or a plurality of helicases. The helicase orhelicases in the preparation may be selected from the class of 3′ to 5′helicases or the class of 5′ to 3′ helicases. More particularly, thehelicase preparation may include a helicase from superfamily 1-4 or anAAA⁺ helicase. The helicase may be a hexameric helicase or a monomericor dimeric helicase. More particularly, the helicase may be a UvrDhelicase or homolog thereof, for example a thermostable helicase orhomolog thereof.

In additional embodiments of the invention, the helicase preparation mayinclude one or more helicases selected from the group consisting of: E.coli UvrD helicase, Tte-UvrD helicase, T7 Gp4 helicase, RecBCD helicase,DnaB helicase, MCM helicase, Rep helicase, RecQ helicase, PcrA helicase,SV40 large T antigen helicase, Herpes virus helicase, yeast Sgs1helicase, DEAH_ ATP-dependent helicases and Papillomavirus helicase E1protein and homologs thereof.

Additionally, the helicase preparation includes a nucleotidetriphosphate (NTP) or deoxynucleotide triphosphate (dNTP) for example,adenosine triphosphate (ATP), deoxythymidine triphosphate (dTTP) ordeoxyadenosine triphosphate (dATP). A suitable concentration for theenergy source is in the range of about 0.1-50 mM.

In additional embodiments of the invention, the helicase preparationincludes a single strand binding protein, for example, T4 gene 32 SSB,E. coli SSB, T7 gene 2.5 SSB, phage phi29 SSB and derivatives therefromand an accessory protein for example, MutL.

Embodiments of the invention include detecting pathogens in biologicalsamples by helicase dependent amplification, where the target nucleicacid is a nucleic acid from the pathogen. Alternatively, sequencevariations in chromosomal DNA can be determined when the target nucleicacid is a fragment of chromosomal DNA. This approach can be used todetect single nucleotide polymorphisms in the target nucleic acid fromdifferent sources.

In an embodiment of the invention, a kit is provided that includes ahelicase preparation and a nucleotide triphosphate or deoxynucleotidetriphosphate and a DNA polymerase and instructions for performinghelicase dependent amplification. The kit may be used for example in thefield, in the laboratory with standard equipment, or for high throughputscreening of samples.

In an embodiment of the invention, a method is provided for determiningwhether a helicase for use in a helicase preparation is suited forexponentially and selectively amplifying a target nucleic acid, whichincludes the steps of: preparing a helicase preparation comprising thehelicase, an NTP or dNTP, a buffer, wherein the buffer of Tris-acetateor Tris-HCl providing a pH in the range of about pH 6.0-9.0, and aconcentration of NaCl or KCl in a concentration range of 0-200 mM andoptionally a single stranded binding protein and/or an accessoryprotein; adding a target nucleic acid in varying concentrations or copynumber, oligonucleotide primers, four dNTPs and a DNA polymerase to thehelicase preparation; incubating the mixture at a temperature betweenabout 20° C. and 75° C.; and analyzing the amplified DNA to determinewhether selective and exponential amplification has occurred.

Composition of the reaction mixture, conditions of the reaction andconcentration of the reactants can be varied within certain rangesprovided herein to identify the optimum conditions for helicasedependent amplification.

An embodiment of the invention includes a method for identifying an RNAmolecule that includes: (a) synthesizing a cDNA from the RNA molecule byRT to form an RNA/DNA duplex; (b) at least partially separating theduplex into a cDNA and an RNA using a thermostable helicase; (c)amplifying the cDNA; and (d) characterizing the cDNA to determine theidentity of the RNA molecule. In particular examples of the method, RTis achieved by means of a reverse transcriptase or a DNA polymerase withRT activity. In addition or alternatively, amplification of the cDNA maybe achieved by helicase-dependent amplification (HDA), loop-mediatedisothermal amplification, rolling cycle amplification,strand-displacement amplification (SDA) and polymerase chain reaction(PCR).

In an embodiment of the invention, the RNA in the DNA/RNA duplex can bereused for RT and amplification in a repeating cycle. The entire methodcan be achieved in a single buffer in a single reaction vessel oralternatively can be achieved using separate buffers for some or all ofthe steps. For example, steps (a) and (b) can be performed in a singlebuffer or in different buffers which may differ from the buffer orbuffers used for steps (b) and (c).

In an embodiment of the method, a single strand binding protein such asdescribed in U.S. patent application 2004-0058378 is not added in step(b) or (c) above. Accordingly, the method may utilize a helicase in areaction mixture that is essentially free of a single strand bindingprotein. A thermostable helicase may be selected that has an effectiveactivity in the absence of a single strand binding protein.

In an embodiment of the invention, a method is provided forexponentially and selectively amplifying a target nucleic acid thatincludes: (a) providing single strand templates of the target nucleicacid to be amplified; (b) adding oligonucleotide primers for hybridizingto the templates of step (a); (c) synthesizing an extension product ofthe oligonucleotide primers which are complementary to the templates, bymeans of a DNA polymerase to form a duplex; (d) contacting the duplex ofstep (c) with a thermostable helicase for unwinding the duplex; and (e)repeating steps (b)-(d) to exponentially and selectively amplify thetarget nucleic acid. In an example of the method, the thermostablehelicase is utilized in the absence of a single strand binding protein.

In an embodiment of the invention, a kit is provided that includes athermostable helicase, a reverse transcriptase, a DNA polymerase and abuffer containing at least three reagents selected from the groupconsisting of: a magnesium salt, a sodium salt, dNTP, and dATP; the kitincluding instructions for use in amplifying a DNA in a reaction thatdoes not include a single strand binding protein. The buffer may forexample have a pH in the range of pH 8-pH 10. In other examples of thekit, the polymerase may be Bst polymerase and/or the thermostablehelicase may be Tte-UvrD helicase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic Diagram of Helicase Displacement Amplification where(1) refers to primers annealing to single strand DNA, (2) refers to DNApolymerase extending the primers, where one duplex is amplified to twoduplexes, and (3) refers to repeating the process to result inexponential amplification.

FIG. 2A. Schematic presentation of HDA amplification of anoligonucleotide with primers for producing an amplified product.

FIG. 2B. An HDA reaction according to FIG. 2A in which the HDA productis characterized on a 3% LMP agarose gel (Lane 1) and Lane 2 containsthe pBR322/MspI ladder used as size marker (M).

FIG. 3. Schematic diagram of selectively amplification of a targetsequence from a large DNA molecule containing that sequence by HDA where(4) is dsDNA separation/Primer annealing, (5) is Primer extension by apolymerase, (6) is unwinding by a Helicase and subsequent Primerannealing, (7) is primer extension by a DNA polymerase and (8) isunwinding, annealing, and extension.

FIG. 4. Amplification of target sequences of various sizes from DNAplasmids.

HDA reactions were performed using an UvrD helicase preparationcontaining E. coli UvrD helicase, E. coli MutL, T4 Gp32 and ATP plus apolymerases, two primers (1224 and 1233), and target DNA of differentlengths in plasmid pAH1. The amplification product was analyzed by gelelectrophoresis on a 3% LMP agarose gel. Lane 1: 110-bp, Lane 2: 200 bp;lane 3: 300 bp; lane 4: 400 bp; lane 5: 650 bp length target DNA. M: 100bp DNA ladder sizing marker.

FIGS. 5A-5B. Amplification of target sequences from bacterial genomicDNA using two different polymerases. Target nucleic acids were amplifiedfrom T. denticola genomic DNA using a UvrD helicase preparationcontaining E. coli UvrD helicase, E. coli MutL, T4 Gp32 and ATP plus twodifferent polymerases. Amplification products were analyzed by gelelectrophoresis on a 3% LMP agarose gel.

FIG. 5A: HDA using exo⁻ Klenow Fragment of DNA polymerase I;

Lane 1: Product of HDA using primer-58861 and primer-58862. Lane 2:Product of HDA with primer-58861 and primer-58863.

FIG. 5B: HDA using T7 sequenase and primers-58861 and 58863: Lane 1: 1.5units of T7 Sequenase; Lane 2: 3.5 units of T7 Sequenase; and Lane Mshows a 100 bp DNA ladder used as sizing marker.

FIG. 6. Amplification of target sequences from human genomic DNA.

HDA reaction was carried out using a helicase preparation containing E.coli UvrD helicase, MutL, T4 Gp32, and ATP plus a DNA polymerases, twoprimers and human genomic DNA. HDA products were analyzed by gelelectrophoresis using a 3% LMP agarose gel. M: 100 bp DNA ladder used assizing marker. HDA product from: 100 ng initial human genomic DNA(Lane 1) from 150 ng initial human genomic DNA (Lane 2), from 200 nginitial human genomic DNA (lane 3).

FIG. 7. Amplification of target sequences coupled to cDNA synthesis (RTamplification).

The HDA reaction was coupled with cDNA synthesis. The first strand cDNA(RNA/DNA hybrid) was further amplified by HDA using a helicasepreparation containing E. coli UvrD helicase, MutL, T4 Gp32, and ATPplus a DNA polymerases, and two primers which are specific to the ratGAPDH gene. The amplification products: 2 μl first cDNA strand (Lane 1),4 μl first cDNA strand (Lane 2) were analyzed by gel electrophoresis ona 3% LMP agarose gel. M: ØX174 DNA-HaeIII DNA ladder.

FIG. 8. Sensitivity of amplification of various copy numbers of targetsequences from bacterial genomic DNA. HDA reactions were carried outusing a helicase preparation containing E. coli UvrD helicase, MutL, T4Gp32, and ATP plus a DNA polymerases, two primers (primer-58861 andprimer-58862), and various amount of Treponema denticola genomic DNA.The amplification products were analyzed by gel electrophoresis on a 3%LMP agarose gel. The number of copies of the single Treponema denticolachromosome initially present in each HDA reaction is shown above eachlane in descending numbers of 10⁷, 10⁶, 10⁵, 10⁴, 10³, 10², 10 and 0.

FIG. 9. Amplification of target sequences from bacterial genomic DNAwithout prior denaturation.

HDA reaction was carried out using a helicase preparation containing E.coli UvrD helicase, MutL, T4 Gp32, and ATP plus a DNA polymerases, twoprimers (primer-58861 and primer-58862), and Treponema denticola genomicDNA. HDA products were analyzed by gel electrophoresis using a 3% LMPagarose gel. M: ØX174 DNA-HaeIII DNA ladder used as sizing marker.

FIG. 10. Amplification of a 2.3-Kb target DNA using a single helicase(T7 Gp4B helicase) or two helicases (UvrD helicase and T7 Gp4Bhelicase).

HDA reactions were performed using two primers (1224 and 1233) andplasmid pCR-Rep in the presence of the T7 Gp4B helicase preparationincluding T7 Gp4B helicase and T7 Gp2.5 SSB (Lane 1): in the presence ofa helicase preparation containing both T7 Gp4B helicase and UvrDhelicase (Lane 2): negative control, no helicase (Lane 3). M: 2-log DNAladder used as sizing marker. The products of HDA are shown by 1% gelelectrophoresis.

FIG. 11. Amplification of 400 bp target DNA using RecBCD helicase.

HDA reactions were performed using a RecBCD helicase preparationcontaining RecB^(D1067A)CD helicase, T4 Gp32 and ATP plus a polymerases(T7 Sequenase), two primers (1224 and 1233), and target DNA. Gelelectrophoresis of HDA products on a 1% agarose gel is shown where Lane2 shows the amplification product from a helicase preparation containingboth RecB^(D1067A)CD helicase and T7 Sequenase and Lane 3 shows anegative control with no helicase. Marker: 2-log DNA ladder used assizing marker (NEB).

FIG. 12. Amplification of a target sequence from bacterial genomic DNAby thermostable HDA.

HDA reaction was carried out using a helicase preparation containing thethermostable Tte-UvrD helicase, T4 Gp32, and dATP plus a thermostableBst DNA polymerases, two primers and T. denticola genomic DNA. Theamplification product was analyzed by gel electrophoresis on a 2% LMPagarose gel. M: 100 bp DNA ladder used as sizing marker (NEB). Lane 1:82 bp product.

FIG. 13. Amplification of a target sequence from the genomic DNA ofNeisseria gonorrhoeae by thermostable helicases absent some or allaccessory proteins.

HDA reaction was carried out using various helicase preparations plus athermostable Bst DNA polymerases, two primers and Neisseria gonorrhoeaegenomic DNA. One helicase preparation contains the thermostable Tte-UvrDhelicase, T4 Gp32, and dATP (Lane 1). The second helicase preparationcontains the thermostable Tte-UvrD helicase and dATP (Lane 2). In acontrol reaction, only T4 Gp 32 and ATP is present in the preparation(Lane 3). M: 100 bp DNA ladder used as sizing marker (NEB).

FIG. 14. Real-time detection of an oral pathogen, T. denticola, by HDAmethod.

HDA reaction was carried out using an UvrD helicase preparationcontaining E. coli UvrD helicase, MutL, T4 Gp32, and ATP plus a DNApolymerases, T. denticola genomic DNA, a fluorescent labeled LUX primer(Invitrogen) and a reverse primer. The amplification product wasdetected in real-time by measuring FAM fluorescent signals using areal-time PCR machine, iCycler, (Bio-Rad). 1 & 2: two identicalreactions in which HDA was performed in the presence of genomic DNA,primers, and the UvrD HDA system. 3: HDA was performed similar to 1 & 2,except the genomic DNA was absent (negative control).

FIG. 15. Sequence of plasmid pAH1 (SEQ ID NO:9).

FIG. 16. Sequence of earRI gene T. denticola (SEQ ID NO:10).

FIG. 17 shows a schematic diagram of an RT-HDA reaction.

Step 1 shows binding a single primer to a target RNA.

Step 2 shows the synthesis of a first strand cDNA by RT of the targetRNA using a reverse transcriptase or polymerase with RT properties.

Step 3 shows the helicase (black triangles) unwinding the DNA/RNA duplexinto at least partial single strand nucleic acids.

Step 4 shows the addition of at least a second primer so that the firstand second primers bind to the boundaries of the nucleic acid sequenceof interest so as to permit amplification of the DNA sequence betweenthe primers.

Step 5-1 shows how displaced single strand RNA or the original targetRNA undergoes an RT reaction (repeating steps 1-4).

Step 5-2 shows how primer binds to single strand DNA (ssDNA) permittingamplification to occur by HDA or other type of amplification.

Steps 6-9 show stages during the amplification.

Step 6 shows how the duplex DNA is unwound either by heat or by HDA orother means.

Step 7 shows primers binding to ssDNA.

Step 8 shows extension of the DNA polymerases from primers.

Step 9 shows that the amplified products enter the next round ofamplification.

Dashed lines: RNA; Solid thick lines: DNA; Lines with arrows: primers.Black triangles: helicases; grey rectangles: reverse transcriptases;grey circles: DNA polymerases.

FIG. 18 shows the results of gel electrophoresis (2% agarose) ofamplified GAPDH RNA in a two-step reaction.

Two-step RT-HDA reaction was performed in two different reaction tubes.The first strand cDNA synthesis was carried out using ProtoScript® Kit(New England Biolabs, Inc., Ipswich, Mass.) and 1 mg of human total RNA.The first-strand cDNA synthesized product was then diluted and amplifiedusing HDA in the presence of Tte-UvrD (helicase) and Bst DNA polymerasein a total volume of 50 ml. Ten ml of each sample was analyzed on thegel. The percentages of the first-strand cDNA synthesized product variedas indicated in lanes 2-7.

Lane 1: 200 ng of Low Molecular Weight DNA Ladder (New England Biolabs,Inc., Ipswich, Mass.);

Lane 2: 1%;

Lane 3: 0.1%;

Lane 4: 0.01%;

Lane 5: 0.001%;

Lane 6: 0.0001%;

Lane 7: 0.

FIG. 19 shows the results of gel electrophoresis (2% agarose) ofamplified GAPDH RNA starting from different amounts of human total RNAin a two-step reaction.

Two-step RT-HDA reaction was performed in one reaction tube. RT-HDA wascarried out by mixing Tte-UvrD, M-MuLV reverse transcriptase, Bst DNApolymerase, and thermophilic HDA (tHDA) buffer in one tube in a totalvolume of 50 ml, incubated at 42° C. for 5 minutes followed by 65° C.for 60 minutes. 10 ml of each sample was analyzed on the gel. Theamounts of total RNA in lanes 1-8 are as follows:

Lanes 1, 2, 3: 100 ng;

Lane 4: 10 ng;

Lane 5: 1 ng;

Lane 6: 100 pg;

Lane 7: 10 pg;

Lane 8: 0;

Lane 9: 200 ng of Low Molecular Weight DNA Ladder (New England Biolabs,Inc., Ipswich, Mass.).

The first two lanes are from control reactions without reversetranscriptase or helicase respectively.

FIG. 20 shows the results of gel electrophoresis (2% agarose) ofamplified GAPDH RNA starting from different amounts of human total RNAin a one-step reaction.

One-step RT-HDA reaction was performed in a single reaction vessel.RT-HDA was carried out by mixing Tte-UvrD, SuperScript™ III (Invitrogen,Carlsbad, Calif.) reverse transcriptase, Bst DNA polymerase, and tHDAbuffer in one tube with a total volume of 50 ml and incubated at 62.5°C. for 60 minutes. 10 _l of each sample was analyzed on the gel. Theamounts of total RNA are:

Lane 1: 200 ng of Low Molecular Weight DNA Ladder (New England Biolabs,Inc., Ipswich, Mass.).

Lane 2: 100 ng;

Lane 3: 10 ng;

Lane 4: 1 ng;

Lane 5: 100 pg;

Lane 6: 0.

FIG. 21 shows the results of gel electrophoresis (2% agarose) ofamplified GAPDH RNA starting from different amounts of human total RNAand using a modified one-step RT-HDA reaction.

RT-HDA was carried out by first denaturing Mix A containing RNA templateand a pair of GAPDH specific primers at 95° C. for 2 minutes. Mix Bcontaining Tte-UvrD, ThermoScript™ (Invitrogen, Calif.) reversetranscriptase, Bst DNA polymerase was then added into Mix A andincubated at 65° C. for 120 minutes. 10 ml was analyzed on the gel. Theamounts of total RNA are:

Lane 1: 250 ng of Low Molecular Weight DNA Ladder (New England Biolabs,Inc., Ipswich, Mass.).

Lane 2: 200 ng;

Lane 3: 20 ng;

Lane 4: 2 ng;

Lane 5: 200 pg;

Lane 6: 20 pg;

Lane 7: 2 pg;

Lane 8: 0.

FIG. 22 shows the detection of Enterovirus using a modified one-stepRT-HDA, varying amounts of Enterovirus RNA and 2% agarose gelelectrophoresis. RT-HDA was carried out as described in FIG. 5 using apair of Enterovirus specific primers. 10 ml was analyzed on the gel. Theamounts of Enterovirus RNA (EV product) are:

Lane 1: 4000 copies;

Lane 2: 400 copies;

Lane 3: 40 copies;

Lane 4: 0.

Lane 5: 250 ng of Low Molecular Weight DNA Ladder (New England Biolabs,Inc., Ipswich, Mass.).

DETAILED DESCRIPTION OF EMBODIMENTS

A novel amplification methodology is described herein which is referredto as “Helicase Dependent Amplification” (HDA). Helicase-DependentAmplification (HDA) is based on the unwinding activity of a DNAhelicase. This novel process uses a helicase rather than heat toseparate the two strands of a DNA duplex generating single-strandedtemplates for the purpose of in vitro amplification of a target nucleicacid. Sequence-specific primers hybridize to the templates and are thenextended by DNA polymerases to amplify the target sequence. This processrepeats itself so that exponential amplification can be achieved at asingle temperature (FIG. 1).

This amplification system has improved characteristics overamplification procedures described in the prior art. These improvementsinclude for example, the ability to amplify long target sequences ofnucleic acids isothermally with high fidelity.

HDA relies on one or more helicases to separate (melt, or unwind) twostrands of a nucleic acid duplex. HDA further utilizes a DNA or RNApolymerase to extend primers which are hybridized to single strandednucleotide sequences to form complementary primer extension products.This process repeats itself so that exponential amplification can beachieved at a single temperature. Some advantages of the presentembodiments over amplification procedures in the prior art include theability to isothermally amplify long target sequences of DNA and RNA(longer than about 200 nucleotides more particularly, greater than about500 nucleotides, more particularly greater than about 1000 nucleotides,more particularly, greater than 2000 nucleotides, more particularly upto about 50,000 nucleotides, more particularly as much as about 100,000nucleotides) and the ability to amplify target sequences at onetemperature from the beginning to the end.

Definitions

For convenience, certain terms employed in the specification, examplesand appended claims are collected here.

The term “Nucleic acid” refers to double stranded or single strandedDNA, RNA molecules or DNA/RNA hybrids. Those molecules which are doublestranded nucleic acid molecules may be nicked or intact. The doublestranded or single stranded nucleic acid molecules may be linear orcircular. The duplexes may be blunt ended or have single stranded tails.The single stranded molecules may have secondary structure in the formof hairpins or loops and stems. The nucleic acid may be isolated from avariety of sources including the environment, food, agriculture,fermentations, biological fluids such as blood, milk, cerebrospinalfluid, sputum, saliva, stool, lung aspirates, swabs of mucosal tissuesor tissue samples or cells. Nucleic acid samples may obtained from cellsor viruses and may include any of: chromosomal DNA, extra chromosomalDNA including plasmid DNA, recombinant DNA, DNA fragments, messengerRNA, transfer RNA, ribosomal RNA, double stranded RNA or other RNAs thatoccur in cells or viruses. The nucleic acid may be isolated, cloned orsynthesized in vitro by means of chemical synthesis. Any of the abovedescribed nucleic acids may be subject to modification where individualnucleotides within the nucleic acid are chemically altered (for example,by methylation). Modifications may arise naturally or by in vitrosynthesis. The term “duplex” refers to a nucleic acid molecule that isdouble stranded in whole or part.

The term “target nucleic acid” refers to a whole or part of nucleic acidto be selectively amplified and which is defined by 3′ and 5′boundaries. The target nucleic acid may also be referred to as afragment or sequence that is intended to be amplified. The size of thetarget nucleic acid to be amplified may be, for example, in the range ofabout 50 bp to about 100 kb including a range of above 100-5000 bp. Thetarget nucleic acid may be contained within a longer double stranded orsingle stranded nucleic acid. Alternatively, the target nucleic acid maybe an entire double stranded or single stranded nucleic acid.

The terms “melting”, “unwinding” or “denaturing” refer to separating allor part of two complementary strands of a nucleic acid duplex.

The term of “hybridization” refers to binding of an oligonucleotideprimer to a region of the single-stranded nucleic acid template underthe conditions in which primer binds only specifically to itscomplementary sequence on one of the template strands, not other regionsin the template. The specificity of hybridization may be influenced bythe length of the oligonucleotide primer, the temperature in which thehybridization reaction is performed, the ionic strength, and the pH.

The term “primer” refers to a single stranded nucleic acid capable ofbinding to a single stranded region on a target nucleic acid tofacilitate polymerase dependent replication of the target nucleic acid.

The term “accessory protein” refers to any protein capable ofstimulating helicase activity. For example, E. coli MutL protein is anaccessory protein (Yamaguchi et al. J. Biol. Chem. 273:9197-9201 (1998);Mechanic et al., J. Biol. Chem. 275:38337-38346 (2000)) for enhancingUvrD helicase melting activity. In embodiments of the method, accessoryproteins are desirable for use with selected helicases. In alternativeembodiments, unwinding of nucleic acids may be achieved by helicases inthe absence of accessory proteins.

The term “cofactor” refers to small-molecule agents that are requiredfor the helicase unwinding activity. Helicase cofactors includenucleoside triphosphate (NTP) and deoxynucleoside triphosphate (dNTP)and magnesium (or other divalent cations). For example, ATP (adenosinetriphosphate) may be used as a cofactor for UvrD helicase at aconcentration in the range of 0.1-100 mM and preferably in the range of1 to 10 mM (for example 3 mM). Similarly, dTTP (deoxythymidinetriphosphate) may be used as a cofactor for T7 Gp4B helicase in therange of 1-10 mM (for example 3 mM).

The term “helicase” refers here to any enzyme capable of unwinding adouble stranded nucleic acid enzymatically. For example, helicases areenzymes that are found in all organisms and in all processes thatinvolve nucleic acid such as replication, recombination, repair,transcription, translation and RNA splicing. (Kornberg and Baker, DNAReplication, W.H. Freeman and Company (2^(nd) ed. (1992)), especiallychapter 11). Any helicase that translocates along DNA or RNA in a 5′ to3′ direction or in the opposite 3′ to 5′ direction may be used inpresent embodiments of the invention. This includes helicases obtainedfrom prokaryotes, viruses, archaea, and eukaryotes or recombinant formsof naturally occurring enzymes as well as analogues or derivativeshaving the specified activity. Examples of naturally occurring DNAhelicases, described by Kornberg and Baker in chapter 11 of their book,DNA Replication, W.H. Freeman and Company (2^(nd) ed. (1992)), includeE. coli helicase I, II, III, & IV, Rep, DnaB, PriA, PcrA, T4 Gp41helicase, T4 Dda helicase, T7 Gp4 helicases, SV40 Large T antigen, yeastRAD. Additional helicases that may be useful in HDA include RecQhelicase (Harmon and Kowalczykowski, J. Biol. Chem. 276:232-243 (2001)),thermostable UvrD helicases from T. tengcongensis (disclosed in thisinvention, Example XII) and T. thermophilus (Collins and McCarthy,Extremophiles. 7:35-41. (2003)), thermostable DnaB helicase from T.aquaticus (Kaplan and Steitz, J. Biol. Chem. 274:6889-6897 (1999)), andMCM helicase from archaeal and eukaryotic organisms ((Grainge et al.,Nucleic Acids Res. 31:4888-4898 (2003)).

Examples of helicases for use in present embodiments may also be foundat the following web address: http://blocks.fhcrc.org (Get Blocks byKeyword: helicase). This site lists 49 Herpes helicases, 224 DnaBhelicases, 250 UvrD-helicases and UvrD/Rep helicases, 276DEAH_ATP-dependent helicases, 147 Papillom_E1 Papillomavirus helicase E1protein, 608 Viral helicase1 Viral (superfamily 1) RNA helicases and 556DEAD_ ATP-dependent helicases. Examples of helicases that generallyreplicate in a 5′ to 3′ direction are T7 Gp4 helicase, DnaB helicase andRho helicase, while examples of helicases that replicate in the 3′-5′direction include UvrD helicase, PcrA, Rep, NS3 RNA helicase of HCV.

In an embodiment of the invention, the helicase is provided in a“helicase preparation”. The helicase preparation refers to a mixture ofreagents which when combined with a DNA polymerase, a nucleic acidtemplate, four deoxynucleotide triphosphates, and primers are capable ofachieving isothermal, exponential and specific nucleic acidamplification in vitro.

More particularly, the helicase preparation includes a helicase, anenergy source such as a nucleotide triphosphate (NTP) or deoxynucleotidetriphosphate (dNTP), and a single strand DNA binding protein (SSB). Oneor more additional reagents may be included in the helicase preparation,where these are selected from the following: one or more additionalhelicases, an accessory protein, small molecules, chemical reagents anda buffer.

Where a thermostable helicase is utilized in a helicase preparation, thepresence of a single stranded binding protein is optional.

The term “HDA system” has been used herein to describe a group ofinteracting elements for performing the function of amplifying nucleicacids according to the Helicase-Dependent Amplification method describedherein. The HDA system includes an helicase preparation, a polymeraseand optionally a topoisomerase.

For example, the UvrD HDA system may be constituted by mixing together,a UvrD helicase preparation (for example, an E. coli UvrD helicasepreparation or a Tte-UvrD helicase preparation) and a DNA polymerasesuch as Exo⁻ Klenow Fragment, DNA polymerase Large fragment, Exo⁺ KlenowFragment or T7 Sequenase.

Another example is the T7 HDA system which includes a T7 helicasepreparation (T7 Gp4B helicase, T7 Gp2.5 SSB, and dTTP), and T7Sequenase.

Another example is RecBCD HDA system which includes a RecBCD preparation(RecBCD helicase with T4gp 32) and T7 Sequenase.

Any selected HDA system may be optimized by substitution, addition, orsubtraction of elements within the mixture as discussed in more detailbelow.

The term “HDA” refers to Helicase Dependent Amplification which is an invitro method for amplifying nucleic acids by using a helicasepreparation for unwinding a double stranded nucleic acid to generatetemplates for primer hybridization and subsequent primer-extension. Thisprocess utilizes two oligonucleotide primers, each hybridizing to the3′-end of either the sense strand containing the target sequence or theanti-sense strand containing the reverse-complementary target sequence.The HDA reaction is a general method for helicase-dependent nucleic acidamplification.

“Isothermal amplification” refers to amplification which occurs at asingle temperature. This does not include the single brief time period(less than 15 minutes) at the initiation of amplification which may beconducted at the same temperature as the amplification procedure or at ahigher temperature.

Helicases use the energy of nucleoside triphosphate (for example ATP)hydrolysis to break the hydrogen bonds that hold the strands together induplex DNA and RNA (Kornberg and Baker, DNA Replication, W.H. Freemanand Company (2^(nd) ed. (1992)), especially chapter 11). Helicases areinvolved in every aspect of nucleic acid metabolism in the cell such asDNA replication, DNA repair and recombination, transcription, and RNAprocessing. This widespread usage may be reflected by the large numbersof helicases found in all living organisms.

Helicases have been classified according to a number of differentcharacteristics. For example, a feature of different helicases is theiroligomeric structure including helicases with single or multimericstructures. For example, one family of helicases is characterized byhexameric structures while another family consists of monomeric ordimeric helicases.

Another characteristic of helicases is the occurrence of conservedmotifs. All helicases have the classical Walker A and B motifs,associated with ATP-binding and Mg²⁺-binding (reviewed in Caruthers andMcKay. Curr. Opin. Struct. Biol. 12:123-133 (2002), Soultanas andWigley. Trends Biochem. Sci. 26:47-54 (2001)). Helicases have beenclassified into several superfamilies (Gorbalenya and Koonin. Curr.Opin. Struct. Biol. 3:419-429 (1993)) according to the number ofhelicase signature motifs and differences in the consensus sequences formotifs. superfamilies 1 and 2 have seven characteristic helicasesignature motifs and include helicases from archaea, eubacteria,eukaryotes and viruses, with helicases unwinding duplex DNA or RNA ineither 3′ to 5′ direction or 5′ to 3′ direction. Examples of superfamily1 helicases include the E. coli UvrD helicase, the T. tengcongensis UvrDhelicase, and the B subunit of RecBCD. Superfamily 3 has three motifsand superfamily 4 has five motifs. Examples of superfamily 4 helicasesinclude the T7 Gp4 helicase and DnaB helicases. A new family differentfrom those canonical helicases is the AAA⁺ family (the extended familyof ATPase associated with various cellular activities).

A third type of classification relates to the unwinding directionalityof helicases i.e. whether the helicase unwinds the nucleic acid duplexin a 5′-3′ direction (such as T7 Gp4 helicase) or in a 3′-5′ direction(such UvrD helicase) based on the strand on which the helicase binds andtravels.

A fourth type of classification relates to whether a helicase preferablyunwinds blunt ended nucleic acid duplexes or duplexes with forks orsingle stranded tails. Blunt-ended nucleic acid duplexes may not berequired in the first cycle of helicase-dependent amplification but aredesirable in subsequent cycles of amplification because along with theprogress of the amplification reaction the blunt-ended target fragmentbecomes the dominant species (FIG. 3). These blunt-ended target nucleicacids form template substrates for subsequent rounds of amplification(FIG. 3).

To accomplish HDA described herein, a helicase classified according toany of the above is suitable for nucleic acid amplification. Indeed,Examples II-IX, X, XI and XII demonstrate a sample of the diversity ofhelicases that can be used according to the present methods to achievehelicase dependent amplification.

Example I describes a UvrD helicase preparation. UvrD helicase is asingle-stranded DNA dependent ATPase activity that results in unwindingwith a 3′ to 5′ polarity (Matson, J. Biol. Chem. 261:10169-10175 (1986))and is involved in both DNA repair and recombination. In vivo, UvrDinteracts with a second protein, MutL. MutL is the master coordinator ofthe mismatch repair pathway and dramatically stimulates the unwindingreaction catalyzed by UvrD (See, for example, Yamaguchi et al., J. Biol.Chem. 273, 9197-9201 (1998); Mechanic et al., J. Biol. Chem. 275,38337-38346 (2000)). Examples XII and XIII show that an accessoryprotein is not always necessary in an optimized helicase preparationalthough it may preferably be used for some helicases such as UvrD. Therequirement of an accessory protein for a particular helicase in HDA canbe readily determined using an assay such as described in Examples II toV and analyzing the HDA product by gel electrophoresis.

E. coli UvrD helicase is a superfamily 1 helicase. E. coli UvrD helicaseis able to unwind blunt-ended DNA duplexes as well as nicked circularDNA molecules (Runyon and Lohman, J. Biol. Chem. 264:17502-17512(1989)). At low concentrations of UvrD, optimum unwinding requires a3′-single stranded DNA tail but at higher concentrations, the unwindingcan be initiated at nicks or blunt ends (Runyon, et al., Proc. Natl.Acad. Sci. USA 87:6383-6387 (1990)).

In another example of HDA, T7 gene 4 protein is used in a helicasepreparation to amplify a target nucleic acid. T7 gene 4 protein is ahexameric replicative helicase which contains both a primase activityand a 3′ to 5′ helicase activity (Lechner and Richardson, J. Biol. Chem.258:11185-11196 (1983)). The amino-terminal truncated version of gene 4protein, T7 gene 4B protein (T7 Gp4B helicase), only contains DNAhelicase activity. The cloning and purification of the T7 Gp4B helicasehas been described by Bernstein and Richardson (J. Biol. Chem.263:14891-14899 (1988)). T7 gene 2.5 protein is a single strand DNAbinding protein and it stimulates T7 DNA polymerase activity (Kim etal., J. Biol. Chem. 267:15032-15040 (1992)). The preparation of T7 Gp2.5SSB has been described previously (Kim et al., J. Biol. Chem.267:15022-5031 (1992)).

In another example of HDA, E. coli RecBCD protein is used in helicasepreparation. E. coli RecBCD is a protein complex containing onesuperfamily 1 helicase (RecB) and one 5′ to 3′ helicase (RecD), is usedto amplify a target fragment. E. coli RecBCD helicase is a trimeric,multifunctional enzyme, which is both an ATP-dependent helicase and aDNA nuclease (Roman and Kowalczykowski, Biochemistry. 28:2863-2873(1989)). The RecB subunit possesses a 3′ to 5′ DNA helicase activity andalso an exonuclease activity. The exonuclease activity can be abolishedby site directed mutagenesis resulting in an exonuclease deficientRecB^(D1067A)CD which is able to unwind duplex DNA without degradation(Wang et al., J. Biol. Chem. 275, 507-513 (2000)). RecD protein is alsoa DNA helicase which possesses a 5′ to 3′ polarity (Taylor and Smith,Nature 423, 889-893 (2003)). RecB and RecD helicases are both active inintact RecBCD via a bipolar translocation model. The two DNA helicasesare complementary, travel with opposite polarities, but in the samedirection, on each strand of the antiparallel DNA duplex. This bipolarmotor organization helps to explain its exceptionally high speed(500-1000 bp/sec) and processivity (>30 kb per binding event);Dillingham et al., Nature 423, 893-897 (2003)).

In another example of HDA, a hexameric replicative helicase, T7 Gp4helicase, is used in a helicase preparation to amplify a target fragmentlonger than one kb. T7 Gp4 helicase belongs to superfamily 4 whosemember including several hexameric helicase such as DnaB and T4 Gp4 andthese helicases have rapid unwinding rates and a high degree ofprocessivity. These helicases recognize single-stranded tails at theborder of duplex region for unwinding. For example, in the presence of aDNA polymerase, E. coli DnaB helicase unwinds DNA at a rate of 750bp/sec with a processivity greater than 50 kb and T7 gp4 helicaseunwinds DNA at a rate of 300 bp/sec with high processivity (Kornberg andBaker, supra (1992)). SV40 large T antigen unwinds DNA at a rate of 75to 100 bp/sec with high processivity (Kornberg and Baker, supra (1992);Li et al., Nature. 423:512-518 (2003)).

While not wishing to be bound by theory, it is possible that althoughsome helicases, such as T7 Gp4, prefer duplex DNA with single-strandedtails, they may still have low unwinding activity on blunt-end duplexDNA molecules. It is also possible that single-stranded tails may betransiently present at the border of a duplex DNA through “terminalbreathing” of the duplex DNA molecule (Roychoudhury et al., Nucleic acidRes. 6:1323-3123 (1979)). These transient single-stranded tails may becaptured by the T7 helicase, which then continues the unwinding process.

Regardless of the source of the target nucleic acid, a helicasepreparation may be used to replace a heat denaturation step duringamplification of a nucleic acid by unwinding a double stranded moleculeto produce a single stranded molecule for polymerase dependentamplification without a change in temperature of reaction. Hencethermocycling that is required during standard PCR amplification usingTaq polymerase may be avoided.

In general, the temperature of denaturation suitable for permittingspecificity of primer-template recognition and subsequent annealing mayoccur over a range of temperatures, for example 20° C.-75° C. Apreferred denaturation temperature may be selected according to whichhelicase is selected for the melting process. Tests to determine optimumtemperatures for amplification of a nucleic acid in the presence of aselected helicase can be determined by routine experimentation byvarying the temperature of the reaction mixture and comparingamplification products using gel electrophoresis.

Denaturation of nucleic acid duplexes can be accelerated by using athermostable helicase preparation under incubation conditions thatinclude higher temperature for example in a range of 45° C.-75° C.(Example XII). Performing HDA at high temperature using a thermostablehelicase preparation and a thermostable polymerase may increase thespecificity of primer binding which can improve the specificity ofamplification.

In certain circumstances, it may be desirable to utilize a plurality ofdifferent helicase enzymes in an amplification reaction. The use of aplurality of helicases may enhance the yield and length of targetamplification in HDA under certain conditions where different helicasescoordinate various functions to increase the efficiency of the unwindingof duplex nucleic acids. For example, a helicase that has lowprocessivity but is able to melt blunt-ended DNA may be combined with asecond helicase that has great processivity but recognizessingle-stranded tails at the border of duplex region for the initiationof unwinding (Example X). In this example, the first helicase initiallyseparates the blunt ends of a long nucleic acid duplex generating 5′ and3′ single-stranded tails and then dissociates from that substrate due toits limited processivity. This partially unwound substrate issubsequently recognized by the second helicase that then continues theunwinding process with superior processivity. In this way, a long targetin a nucleic acid duplex may be unwound by the use of a helicasepreparation containing a plurality of helicases and subsequentlyamplified in a HDA reaction.

Primers

Generally, primer pairs suitable for use in HDA are short syntheticoligonucleotides, for example, having a length of more than 10nucleotides and less than 50 nucleotides. Oligonucleotide primer designinvolves various parameters such as string-based alignment scores,melting temperature, primer length and GC content (Kampke et al.,Bioinformatics 17:214-225 (2003)). When designing a primer, one of theimportant factors is to choose a sequence within the target fragmentwhich is specific to the nucleic acid molecule to be amplified. Theother important factor is to decide the melting temperature of a primerfor HDA reaction. The melting temperature of a primer is determined bythe length and GC content of that oligonucleotide. Preferably themelting temperature of a primer is should about 10 to 30° C. higher thanthe temperature at which the hybridization and amplification will takeplace. For example, if the temperature of the hybridization andamplification is set at 37° C. when using the E. coli UvrD helicasepreparation, the melting temperature of a pair of primers designed forthis reaction should be in a range between about 47° C. to 67° C. If thetemperature of the hybridization and amplification is 60° C., themelting temperature of a pair of primers designed for that reactionshould be in a range between 65° C. and 90° C. To choose the best primerfor a HDA reaction, a set of primers with various melting temperaturescan be tested in a parallel assays. More information regarding primerdesign is described by Kampke et al., Bioinformatics 17:214-225 (2003).

Each primer hybridizes to each end of the target nucleic acid and may beextended in a 3′ to 5′ direction by a polymerase using the targetnucleotide sequence as a template (FIG. 3). Conditions of hybridizationare standard as described in “Molecular Cloning and Laboratory Manual”2^(nd) ed. Sambrook, Rich and Maniatis, pub. Cold Spring Harbor (2003).To achieve specific amplification, a homologous or perfect match primeris preferred. However, primers may include sequences at the 5′ end whichare non complementary to the target nucleotide sequence(s).Alternatively, primers may contain nucleotides or sequences throughoutthat are not exactly complementary to the target nucleic acid. Primersmay represent analogous primers or may be non-specific or universalprimers for use in HDA as long as specific hybridization can be achievedby the primer-template binding at a predetermined temperature.

The primers may include any of the deoxyribonucleotide bases A, T, G orC and/or one or more ribonucleotide bases, A, C, U, G and/or one or moremodified nucleotide (deoxyribonucleotide or ribonucleotide) wherein themodification does not prevent hybridization of the primer to the nucleicacid or elongation of the primer or denaturation of double strandedmolecules. Primers may be modified with chemical groups such asphosphorothioates or methylphosphonates or with non nucleotide linkersto enhance their performance or to facilitate the characterization ofamplification products.

To detect amplified products, the primers may be subject tomodification, such as fluorescent or chemiluminescent-labeling, andbiotinylation (for example, fluorescent tags such as amine reactivefluorescein ester of carboxyfluorescein-Glen Research, Sterling, Va.).Other labeling methods include radioactive isotopes, chromophores andligands such as biotin or haptens which while not directly detectablecan be readily detected by reaction with labeled forms of their specificbinding partners e.g. avidin and antibodies respectively.

Primers as described herein can be prepared by methods known in the art.(see, for example U.S. Pat. No. 6,214,587).

In embodiments, a pair of two sequence-specific primers, one hybridizingto the 5′-border of the target sequence and the other hybridizing to the3′-border of the target (FIG. 3), are used in HDA to achieve exponentialamplification of a target sequence. This approach can be readilydistinguished from Lee et al. (J. Mol. Biol. 316:19-34 (2002)). Multiplepairs of primers can be utilized in a single HDA reaction for amplifyingmultiple targets simultaneously using different detection tags in amultiplex reaction. Multiplexing is commonly used in SNP analysis and indetecting pathogens (Jessing et al., J. Clin. Microbiol. 41:4095-4100(2003)).

Polymerases

Polymerases are selected for HDA on the basis of processivity and stranddisplacement activity. Subsequent to melting and hybridization with aprimer, the nucleic acid is subjected to a polymerization step. A DNApolymerase is selected if the nucleic acid to be amplified is DNA. Whenthe initial target is RNA, a reverse transcriptase is used first to copythe RNA target into a cDNA molecule and the cDNA is then furtheramplified in HDA by a selected DNA polymerase (Example VII). The DNApolymerase acts on the target nucleic acid to extend the primershybridized to the nucleic acid templates in the presence of four dNTPsto form primer extension products complementary to the nucleotidesequence on the nucleic acid template (FIG. 1 and FIG. 3).

The DNA polymerase is selected from a group of polymerases lacking 5′ to3′ exonuclease activity and which additionally may optionally lack 3′-5′exonuclease activity.

Examples of suitable DNA polymerases include an exonuclease-deficientKlenow fragment of E. coli DNA polymerase I (New England Biolabs, Inc.(Beverly, Mass.)), an exonuclease deficient T7 DNA polymerase(Sequenase; USB, (Cleveland, Ohio)), Klenow fragment of E. coli DNApolymerase I (New England Biolabs, Inc. (Beverly, Mass.)), Largefragment of Bst DNA polymerase (New England Biolabs, Inc. (Beverly,Mass.)), KlenTaq DNA polymerase (AB Peptides, (St Louis, Mo.)), T5 DNApolymerase (U.S. Pat. No. 5,716,819), and Pol III DNA polymerase (U.S.Pat. No. 6,555,349). DNA polymerases possessing strand-displacementactivity, such as the exonuclease-deficient Klenow fragment of E. coliDNA polymerase I, Bst DNA polymerase Large fragment, and Sequenase, arepreferred for Helicase-Dependent Amplification. T7 polymerase is a highfidelity polymerase having an error rate of 3.5×10⁵ which issignificantly less than Taq polymerase (Keohavong and Thilly, Proc.Natl. Acad. Sci. USA 86, 9253-9257 (1989)). T7 polymerase is notthermostable however and therefore is not optimal for use inamplification systems that require thermocycling. In HDA, which can beconducted isothermally, T7 Sequenase is a one of the preferredpolymerases for amplification of DNA.

Single-Stranded DNA Binding Proteins

Mesophilic helicases show improved activity in the presence ofsingle-strand binding proteins (SSB). In these circumstances, the choiceof SSB is generally not limited to a specific protein. Examples ofsingle strand binding proteins are T4 gene 32 protein, E. coli SSB, T7gp2.5 SSB, phage phi29 SSB (Kornberg and Baker, supra (1992)) andtruncated forms of the aforementioned.

Other Chemical Reagents

In addition to salt and pH, other chemical reagents, such asdenaturation reagents including urea and dimethyl-sulfoxide (DMSO) canbe added to the HDA reaction to partially denature or de-stabilize theduplex DNA. HDA reactions can be compared in different concentrations ofdenaturation reagents with or without SSB protein. In this way, chemicalcompounds can be identified which increase HDA efficiency and/orsubstitute for SSB in single-strand (ss) DNA stabilization. Most of thebiomacromolecules such as nucleic acids and proteins are designed tofunction and/or form their native structures in a living cell at muchhigh concentrations than in vitro experimental conditions. Polyethyleneglycol (PEG) has been used to create an artificial molecular crowdingcondition by excluding water and creating electrostatic interaction withsolute polycations (Miyoshi, et al., Biochemistry 41:15017-15024(2002)). When PEG (7.5%) is added to a DNA ligation reaction, thereaction time is reduced to 5 min (Quick Ligation Kit, New EnglandBiolabs, Inc. (Beverly, Mass.)). PEG has also been added into helicaseunwinding assays to increase the efficiency of the reaction (Dong, etal., Proc. Natl. Acad. Sci. USA 93:14456-14461 (1996)). PEG or othermolecular crowding reagents on HDA may increase the effectiveconcentrations of enzymes and nucleic acids in HDA reaction and thusreduce the reaction time and amount of protein concentration needed forthe reaction.

Cofactors

ATP or TTP is a commonly preferred energy source for highly processivehelicases. On average one ATP molecule is consumed by a DNA helicases tounwind 1 to 4 base pairs (Kornberg and Baker, supra (1992)). In anembodiment of the invention, the UvrD-based HDA system had an optimalinitial ATP concentration of 3 mM. To amplify a longer target, more ATPmay be consumed as compared to a shorter target. In these circumstances,it may be desirable to include a pyruvate kinase-based ATP regeneratingsystem for use with the helicase (Harmon and Kowalczykowski, Journal ofBiological Chemistry 276:232-243 (2001)).

Topoisomerase

Topoisomerase can be used in long HDA reactions to increase the abilityof HDA to amplify long target amplicons. When a very long linear DNAduplex is separated by a helicase, the swivel (relaxing) function of atopoisomerase removes the twist and prevents over-winding (Kornberg andBaker, supra (1992)). For example, E. coli topoisomerase I (Fermentas,Vilnius, Lithuania) can be used to relax negatively supercoiled DNA byintroducing a nick into one DNA strand. In contrast, E. coli DNA gyrase(topoisomerase II) introduces a transient double-stranded break into DNAallowing DNA strands to pass through one another (Kornberg and Baker,supra (1992)).

Detection of Amplified Nucleic Acids

Amplified nucleic acid product may be detected by various methodsincluding ethidium-bromide staining and detecting the amplified sequenceby means of a label selected from the group consisting of a radiolabel,a fluorescent-label, and an enzyme. For example HDA amplified productscan be detected in real-time using fluorescent-labeled LUX™ Primers(Invitrogen Corporation, Carlsbad, Calif.) which are oligonucleotidesdesigned with a fluorophore close to the 3′ end in a hairpin structure.This configuration intrinsically renders fluorescence quenchingcapability without separate quenching moiety. When the primer becomesincorporated into double-stranded amplification product, the fluorophoreis dequenched, resulting in a significant increase in fluorescentsignal. Example XIV demonstrates real-time detection of a targetsequence using fluorescent-labeled primers and the HDA method.

Identifying a Helicase which can be Used in HDA

To test whether a helicase can be used in the HDA reaction to amplify atarget nucleic acid, a HDA reaction can be set up as following:

(a) a short double stranded oligonucleotide (less than 100 nucleotides)can be used as the substrate for amplification. Primers are preparedwhich can hybridize to the 5′ and 3′ ends of the oligonucleotide. Thedouble-stranded oligonucleotide is denatured to form single strands in afirst mixture of the primers in a standard Tris acetate buffer (10 mM,pH 7.5) or ThermoPol (New England Biolabs, Inc. (Beverly, Mass.)) bufferand varying amounts of dNTPs or NTPs. The mixture is heated to 95° C.for 10 minutes, 53° C. for 1 minute.

(b) a second mixture is prepared where the second mixture has aconcentration of the helicase to be tested in a HDA buffer with a pHwhich is varied between pH6.0 and pH 9.0. The standard buffer may have aconcentration of NaCl and KCl, each in a concentration range of about0-200 mM. The concentration of the helicase is also varied. A singlestranded binding protein such as T4 gp 32 is added together with a DNApolymerase and 4 dNTPs in a standard amount for use in an amplificationreaction which additionally includes a nucleic acid to be amplified andprimers.

(c) the mixtures are combined and incubated for 2 hours at 37° C. (or ata temperature and then analyzed on a 3% GPG LMP agarose gel.

By performing repeated reactions under the different conditionsdescribed above, the optimal conditions for HDA can be determined for aparticular helicase.

The helicase can then be tested for its ability to amplify plasmid DNA,longer DNA molecules and for amplifying short sequence in genomic DNA asillustrated in the Examples for E. coli UvrD helicases.

Helicase dependent amplification is here demonstrated to be an improvedmethod of nucleic acid amplification for use in a wide variety ofapplications. These include amplification following reversetranscription and quantitative amplification using real time HDA. TheExamples below illustrate how HDA is a sensitive and effective methodfor amplifying nucleic acids having a wide range of sizes. One measureof the sensitivity of the HDA reaction is its capacity to amplify anucleic acid sequences in the range of 10 fold to over 1 billion fold.

Table 1 contains some sample values although these are not intended tobe limiting.

TABLE 1 Amplification rates Starting Substrate Amount End Amount Fold ofAmplification Oligo  5 ng 500 ng  100 Plasmid  25 ng of 2700 bp 500 ngof 5000 100 bp Genomic 100 ng of 3 Mb 300 ng of 1 × 10⁵ DNA 100 bpGenomic  0.1 ng of 3 Mb 300 ng of 1 × 10⁹ DNA 100 bp

Amplification Conditions—Temperature

Although other isothermal nucleic acid amplification methods such asStrand-Displacement Amplification can amplify target at a constanttemperature without thermo-cycling, they do require an initialdenaturation step to generate single-stranded template. An advantage ofembodiments of the method is that both unwinding by helicase andamplification can effectively occur at a single temperature throughoutas demonstrated in Example IX. Alternatively, the temperature is raisedto assist initial unwinding of the target nucleic acid by the helicaseand the amplification then proceeds at a single temperature.

We have shown that HDA can be used in place of PCR for amplification ofreverse transcribed product of RNA (Example VII). In addition, HDA isexpected to be useful for quantitative amplification such as found to beuseful in gene expression studies and environmental analyses.Accordingly, where it is desirable to determine the amounts of a targetnucleic acid, HDA can be utilized in a real time end point assay.Accordingly, HDA may be used to determine the relative amounts ofmessenger RNA in a cell in gene expression studies. For example,calibrated gene expression profiles described in WO 0125473 can begenerated using quantitative helicase dependent amplification or Q-HDA.

An embodiment of the method combines reverse transcription of RNA with ahelicase and a subsequent or concomitant amplification step of theresulting cDNA in a single reaction vessel or in a plurality of reactionvessels. The amplification step is optionally an HDA but mayalternatively be a polymerase chain reaction, a loop-mediated isothermalamplification or a rolling circle amplification. RT-HDA may be performedisothermally at a temperature in the range of about 20° C.-75° C., forexample in a range between 60° C.-65° C. In an embodiment of theinvention, RT-HDA can be performed to detect or quantify RNA where HDAreplaces PCR in amplification protocols such as real time RT-HDA,relative RT-HDA, competitive HDA and comparative RT-HDA.

RNA can be amplified in a reaction or a series of reactions that includeRT, thermostable helicase dependent denaturation and amplification.Where the reaction occurs in a single reaction vessel, it is desirableto utilize a single buffer. This constraint is not applicable when RNAis amplified in a series of reactions that occur in different reactionvessels. An advantage of conducting the reactions in a single reactionvessel is that cDNA copies of an RNA target sequence act as a templatefor DNA amplification at the same time as more cDNA is generated fromRNA liberated from the DNA/RNA duplex by RT.

In one embodiment of the invention, the amplification process is ahelicase-dependent amplification (see U.S. patent application2004/0058378). In this approach, amplification can occur in the presenceof a helicase preparation, which includes at least one helicase, anenergy source such as nucleotide triphosphates and a single strandbinding protein or a thermostable helicase in the absence of a singlestrand binding protein. The helicase preparation is combined with a DNApolymerase, deoxyribonucleotides and primers to give rise to HDA. Wherea thermostable helicase is used, a helicase preparation may not berequired. The thermostable helicase may not require a single strandbinding protein but can be combined with a polymerase,deoxyribonucleotides and primers to affect HDA (see Examples XVII-XXII).

Examples of RNA targets include any RNA obtained from a cell or a virussuch as messenger RNA, transfer RNA, ribosomal RNA, double-stranded RNAor other RNAs that occur in cells or viruses.

A reverse transcriptase suitable for use in embodiments of the presentmethod includes any commercially available enzyme used for reversetranscriptase such as M-MuLV, Avian Myeloblastosis Virus (AMV) reversetranscriptase, a thermostable reverse transcriptase such as(SuperScript™, ThermoScript™ (Invitrogen, Carlsbad, Calif.), orTranscriptor (Roche, Basel, Switzerland) reverse transcriptase or apolymerase with reverse transcriptase activity such as Tth polymerase.

Helicases for use in embodiments of the method are enzymes capable ofunwinding double-stranded DNA under normal (mesophilic) or elevatedtemperatures (thermostable). Helicases are abundant in nature and thereis an extensive literature on their properties. They have beenidentified for prokaryotes, eukaryotes and archaea and like otherenzymes, some are thermostable (preferred reaction temperature aboveabout 60° C.) while others are thermolabile (preferred reactiontemperature between about 20° C.-37° C.). An advantage of thermostablehelicases such as Tte-UvrD helicase is that they do not require a singlestrand binding protein, which is required by a thermolabile helicase.While the number of examples that could be provided is extensive,Tte-UvrD helicase is representative of thermostable helicases in generaland teaches a person of ordinary skill in the art how to perform theinvention. Examples of thermolabile helicases include UvrD-likehelicases such as E. coli UvrD helicase, RecBCD helicase, and T7 gene 4helicase and thermostable helicases such as Tte-UvrD helicase (U.S.patent application 20040058378).

Examples of a suitable DNA polymerase include Klenow fragment of E. coliDNA polymerase I, T7 DNA polymerase (Sequenase, USB Corporation,Cleveland, Ohio), Vent® DNA polymerase, Vent® (exo⁻) DNA polymerase (NewEngland Biolabs, Inc., Ipswich, Mass.), Bst DNA polymerase largefragment, and Phusion DNA polymerase (Finnzymes, Espoo, Finland).Preferably, the DNA polymerase lacks 5′ to 3′ exonuclease activity andpossesses strand displacement activity.

The reverse transcriptase, helicase induced denaturation and optionallyan HDA can occur in a single reaction vessel in a single buffer. In oneembodiment, the buffer contains 20 mM Tris-HCl, pH 8.3-8.8, contains 3-5mM MgCl2, 10 mM KCl, 20-40 mM NaCl, 0.4 mM dNTP, and 3 mM dATP or ATP.

The temperature of the reaction is preferably biased towards the optimaltemperature for the helicase reaction. However, if the reversetranscriptase is heat sensitive, the temperature can be modifiedaccordingly.

Performing RT-HDA in a single reaction vessel is preferable. In suchsystem, cDNA copies are generated and amplified from a RNA targetconcurrently using a helicase, a reverse transcriptase, and a DNApolymerase. The first strand cDNA is first synthesized by a reversetranscriptase (SuperScript™, ThermoScript™ (Invitrogen, Carlsbad,Calif.), or Transcriptor (Roche, Basel, Switzerland) reversetranscriptase when the RT and amplification occurs in a single reactionvessel (FIG. 17, steps 1 & 2). The RNA-DNA duplex from the RT is atleast partially unwound by helicases generating single-stranded RNA andDNA templates (FIG. 17, steps 3 & 4). In an embodiment of the invention,the helicase unwinds the full length of the RNA/DNA duplex. Thesingle-stranded RNA enters a next round of RT reaction (FIG. 17, step5-1) generating more first strand cDNA. The ssDNA is converted intodouble-stranded DNA by the DNA polymerase (FIG. 17, step 5-2) andamplified concurrently in the HDA reaction (FIG. 17, steps 6-9). Thisprocess repeats itself to achieve exponential amplification of the RNAtarget sequence (FIG. 17). Helicases that unwind both RNA-DNA duplexesand DNA duplexes are preferred in reactions that occur in a singlereaction vessel. These helicases include a UvrD helicase or homologthereof, for example a thermostable Tte-UvrD helicase.

Alternatively, the RT-HDA can be carried out at two different reactionsin the same or different reaction vessels. For example, the RT may beexecuted at a preferred temperature for the selected reversetranscriptase and the amplification may be preformed at a preferredtemperature for the helicase reaction. An example of a two phasereaction involves making cDNA copies using M-MuLV reverse transcriptaseat for example 42° C. (37° C.-50° C. for RT) for about 3-10 minutes,more particularly 5 minutes, in a first step followed by amplificationin a second step using thermophilic HDA reaction at about 62° C.-66° C.for tHDA, more particularly, about 65° C. for about 60-120 minutes.

Where two different buffer systems are used for RT-HDA, (one for RT andthe other for the amplification step), the first strand cDNA issynthesized by a reverse transcriptase in the presence of either anoligo-dT primer or a sequence-specific primer complimentary to thetarget RNA sequence. In an embodiment of the invention, the RT buffer ina two-phase method is 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10mM DTT. AMV: 50 mM Tris acetate, pH 8.4, 75 mM potassium acetate, 8 mMmagnesium acetate, 10 mM DTT, dNTPs, and target RNA. Aliquots of cDNAcopies from the RT reaction are then transferred into a second reactionbuffer and are subsequently amplified in the presence of a helicase anda polymerase.

The cDNA product can be characterized so as to identify the original RNAsample by methods that are well known in the art such as sequencing anddatabase comparisons, hybridization involving for example DNA arrayssuch as provided on a chip by Nanogen, San Diego, Calif. and anysuitable probe technology including the use of Molecular Beacon andTaqman® technology (Applied Biosystems, Foster City, Calif.).

Real time HDA may be used as a sensitive technique to determine amountsof an organism in a contaminated sample such as E. coli in seawater.Real time detection using sensitive markers such as fluorescence in aHDA reaction has been demonstrated in Example XIV.

HDA may be developed in the context of a compact device for use in fieldactivities and/or laboratory diagnoses. For example, HDA could bepracticed in a microfluidic environment. Microfluidics technologies (labon a chip) are rapidly emerging as key strategies for cost and timesaving by performing biochemical analyses in miniaturized environmentusually at nanoliter scale. Microfluidics technologies have greatpotential to be used as field-portable equipment in pathogen detectionwhen combining with a nucleic acid amplification and detection method.The ability of HDA to amplify nucleic acids in an isothermal conditionwithout initial heat-denaturation makes it a good candidate for thenucleic acid amplification process in a microfluidic device. Similarly,HDA may be used either in kits or in laboratory amplification proceduresto create response profiles of the sort described in InternationalPublication No. WO 0202740 or for monitoring disease (U.S. PublicationNo. 2001018182).

Examples II-XIV illustrate that HDA is effective for amplifying targetnucleic acid from different sources and having different sequences.Examples IV describe amplification of various lengths of targetsequences from DNA plasmids using HDA. Examples X demonstrates thatlonger target sequence (>2 kb) can be amplified by the T7 Gp4B-based HDAsystem. Examples X further demonstrates that the method of usingHelicase-Dependent Amplification to amplify nucleic acids can beperformed using different helicase preparations, such as a helicasepreparation containing T7 Gp4B helicase, or a helicase preparationcontaining more than one helicase, such as T7 Gp4B helicase and UvrDhelicase.

The demonstration in Example VIII that amplification of merely 10 copiesof bacterial genomic DNA can be successfully achieved using HDA,supports the use of HDA for molecular diagnostics application ofinfectious diseases caused by pathogenic bacteria, for example Chlamydiatrachomatis and Neisseria gonorrhoeae. The demonstration that targetsequences can be amplified from human genomic DNA samples (Example VI)supports the use of HDA in identifying genetic alleles corresponding toa particular disease including single nucleotide polymorphisms andforensic applications that rely on characterizing small amounts ofnucleic acid at the scene of a crime or at an archeological site.

The following Examples are provided to aid in the understanding of theinvention and are not construed as a limitation thereof.

The references cited above and below are herein incorporated byreference.

EXAMPLES Example I Cloning and Purifying UvrD Helicase and its AccessoryProtein MUTL

1. Cloning the Genes Encoding UvrD Helicase and MutL Protein

Genes encoding E. coli helicase II or UvrD helicase (Swissprot AccessionNo.: P03018) and its accessory protein E. coli MutL protein (SwissprotAccession No.: P23367) were cloned using the Impact™ system which leadsto a C-terminal translational fusion of a bifunctional tag consisting ofthe S. cerevisiae VMA intein and a chitin-binding domain (New EnglandBiolabs, Inc. (Beverly, Mass.)). This protein purification systemutilizes the DTT-inducible self-cleavage activity of a protein splicingelement (termed an intein) to separate the target protein from theaffinity tag (chitin binding domain). Vent® DNA polymerase was used toamplify UvrD gene from E. coli K12 genomic DNA using primer 5A (5′GGTGGTACCATGGACGTTTCT TACCTGCTC 3′ (SEQ ID NO:1)) and primer 3A (5′GGTGGTGCT CTTCCGCACACCGACTCCAGCCGGGC 3′ (SEQ ID NO:2)). The mutL genewas amplified from E. coli K12 genomic DNA using primer 5B (5′GGTGGTCATATGCCA ATTCAGGTCTTACCG 3′ (SEQ ID NO:3)) and primer 3B (5′GGTGGTTGCTCTTCCGCACTCA TCTTTCAGGGCTTTTATC 3′ (SEQ ID NO:4)). E. ColiK-12 was obtained from New England Biolabs, Inc. (Beverly, Mass.). Thegenomic DNA was isolated with the Qiagen genomic DNA kit (Qiagen, Hilden(Germany)). The primers contained restriction enzymes sites thatallowing the cloning of the mutL gene into the NdeI and SapI sites ofpTYB1 (New England Biolabs, Inc., (Beverly, Mass.)) and the UvrD geneinto the NcoI and SapI sites of pTYB3 (New England Biolabs, Inc.,(Beverly, Mass.)). Ligation products were transformed into ER2502 cells.Positive transformants were screened by selective growth on LB platescontaining 100 μg/ml ampicillin, followed by colony PCR and sequencingof the insert. After analysis of sequencing results, correct constructswere transformed into E. coli ER2566 cells. ER2566 cells containingeither pTYB1-MutL or pTYB3-UvrD were grown at 37° C. in LB mediasupplemented with 100 ug/ml ampicillin. When OD₅₅₀ reached ˜0.5, proteinexpression was induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside(IPTG). After an overnight incubation at 15° C., cells were harvested bycentrifugation.

2. UvrD and MutL Purification

The chitin binding domain (CBD) of the intein tag allowed affinitypurification of the fusion proteins on a chitin bead column (New EnglandBiolabs, Inc., (Beverly, Mass.)). All procedures were performed at 4° C.Cells expressing UvrD from a 6 liter culture were resuspended in 210 mlsonication buffer (20 mM Tris pH 7.8, 0.1 mM EDTA, 50 mM NaCl, 20 μMPMSF, 5% glycerol) and were broken by sonication. The clarified extractwas loaded on a 45-ml chitin bead column pre-equilibrated with 500 ml ofbuffer A (20 mM Tris-HCl (pH 8), 1 mM EDTA) plus 500 mM NaCl. The columnwas washed with 500 ml of buffer A plus 1 M NaCl and 500 ml of buffer Aplus 500 mM NaCl. Induction of self-cleavage was conducted by flushingthe column with 3 column volumes (135 ml) of cleavage buffer (bufferA+500 mM NaCl+50 mM dithiothreitol (DTT)). The cleavage reaction wascarried out at 4° C. for 64 hours in cleavage buffer. The protein waseluted with 67 ml of buffer B (20 mM Tris-HCl (pH 8), 1 mM EDTA, 1 mMDTT) plus 50 mM NaCl. The positive fractions were pooled and loaded on a1 ml-MonoQ column (Pharmacia (Piscataway, N.J.)) which had beenpre-equilibrated with buffer B plus 50 mM NaCl. The flow-through andeluted fractions were analyzed on SDS-PAGE. Helicase activities inpositive fractions were further tested by measuring the ability of thehelicase to displace a fluorescent-labeled oligonucleotide(30-nucleotide, (nt)) from a partial duplex, which was prepared byannealing the 30-nt oligonucleotide to a complementary non-labeled 70-ntoligonucleotide. The displaced 30-nt labeled oligonucleotide was trappedby another non-label 30-nt complementary oligonucleotide. Theoligonucleotides were separated by electrophoresis in a 20%non-denaturing polyacrylamide gel and the displaced oligonucleotideswere visualized by UV light. UvrD protein and helicase activity werefound in the flow-through and the wash fractions. These fractions weremixed and then loaded on a 1-ml Heparin TSK column (Pharmacia(Piscataway, N.J.)). Again, UvrD didn't bind to the column. A one-mlhydroxylapatite column (TosoHaas (Philadelphia, Pa.)) retained UvrD,which eluted at around 340 mM NaCl in a linear gradient (50 mM-1 MNaCl). The pure fractions were pooled and dialyzed overnight againststorage buffer (20 mM Tris-HCl (pH8.2), 200 mM NaCl, 1 mM EDTA, 1 mMEGTA, 15 mM 2-mercaptoethanol, 50% glycerol). The final concentrationwas determined using the Bradford protein assay (Bradford, Anal.Biochem. 72:248-254 (1976)) and SDS polyacrylamide gel electrophoresis(SDS-PAGE).

MutL was purified similarly to UvrD. A 6-liter culture ofER2566/pTYB1-MutL was used. All procedures were performed at 4° C. Thechitin bead column purification conditions were similar to the UvrDexcept that column volume was 14 ml. The column was washed with 125 mlof buffer A plus 1 M NaCl and 125 ml of buffer A plus 500 mM NaCl.Induction of self-cleavage was conducted by flushing the column with 45ml of cleavage buffer (buffer A+500 mM NaCl+50 mM DTT). The cleavagereaction was carried out at 4° C. for 40 hours in cleavage buffer. Theprotein was eluted with 36-ml of buffer B+50 mM NaCl. The positivefractions were pooled and loaded on a 1-ml MonoQ column. MutL was foundin the flow through of the column. The flow-through and the washingfractions were thus pooled and dialyzed against buffer B+40 mM NaCl toget a final NaCl concentration of 50 mM. The sample was loaded on a 1-mlHeparin TSK column. MutL was retained, eluting at 565 mM NaCl. Howeverother protein bands could be detected on SDS-PAGE and an exonucleaseassay showed that exonuclease activity was present in the fractions ofinterest. These fractions were pooled and dialyzed against buffer B+50mM NaCl. The 1-ml MonoQ column was used a second time to separate MutLfrom contaminant proteins. MutL eluted at 220 mM NaCl. The purefractions were pooled and concentrated by a Centriplus YM 10 (Millipore,(Bedford Mass.)) before being dialyzed overnight against the storagebuffer (25 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1 mM 2-mercaptoethanol,0.1 mM EDTA, 50% glycerol). The final concentration was determined usingthe Bradford protein assay and polyacrylamide gel electrophoresis(PAGE).

3. Other Cloning and Purification Systems

In addition to Impact™, helicases and their accessory proteins may bepurified using several alternative methods such as direct cloning(cloning the gene into a vector without an additional tag), His-Tag®(Novagen, Inc. (Madison, Wis.)), and pMAL™ protein fusion & purificationsystem (New England Biolabs, Inc. (Beverly, Mass.)). The E. coli UvrDhelicase was cloned into plasmid pET15b (Novagen, Inc. (Madison, Wis.))and pMAL-c2X (New England Biolabs, Inc. (Beverly, Mass.)). The His-Tagfusion, UvrD-His, was purified using a His.Bind® column and a protocolprovided by the manufactory (Novagen, Inc. (Madison, Wis.)). TheUvrD-His protein was further purified by a hydroxylapatite column. TheMBP-UvrD fusion protein was purified using an amylose column and aprotocol provided by the manufactor (New England Biolabs, Inc.,(Beverly, Mass.)). Both UvrD-His protein and MBP-UvrD fusion proteinshowed functional unwinding activity and could be used in aHelicase-Dependent Amplification reaction.

Example II Method of Amplification of a Nucleic Acid Duplex Target

As a model system for Helicase Dependent Amplification, a synthetic DNAduplex was used as template in the HDA reaction. This exampleillustrates amplification of this DNA duplex using the UvrD HDA system.A method for template denaturation, primer annealing and extension isdescribed below.

Thirty five μl of reaction Component A was made by mixing 10 μl of 5×HDABuffer A (175 mM Tris-HCl (pH7.5), 5 mM DTT), 0.5 μl of 70-bp DNAtemplate derived from top oligodeoxy-nucleotides (2 μM; 5′TGGCTGGTCACCAGAGGGTGGCGCGGAC CGAGTGCGCTCGGCGGCTGCGGAGAGGGGTAGAGCAGGCAGC3′ (SEQ ID NO:5)) and bottom oligodeoxynucleotides (2 μM; 5′GCTGCCTGCTCTACCCCTCTCCGCAGCCGCCGAGCG CACTCGGTC CGCGCCACCCTCTGGTGACCAGCCA3′ (SEQ ID NO:6)), 1 μl of 5′-primer (10 μM; 5′CATGTTAGGTTCTATGGATCGAGTCTGGCTGG TCACCAGAGGG 3′ (SEQ ID NO:7)), 1 μl of3′-primer (10 μM; 5′ TCCCTTAGAGGTCACATTGGATCGAGTCGCTGCCTGCTCTACCCC 3′SEQ ID NO:8)), 10 μl of four dNTPs (2 mM each), 1.5 μl ATP (100 mM), and11 μl dH₂O. The reaction Component A was heated for 2 min at 95° C. todenature the template, 3 min at 53° C. to anneal primers and 2 min at37° C. before adding 0.5 μl of MutL protein (800 ng/μl). Fifteen μl ofreaction Component B was prepared by mixing 10 μl, 5×HDA buffer B (5 mMTris-Cl (pH7.9), 25 mM NaCl, 55 mM MgCl₂, 0.5 mg/ml BSA, 0.5 mM DTT),0.5 μl exo⁻ Klenow Fragment of E. coli DNA polymerase I (5 units/μl),0.5 μl UvrD helicase (200 ng/μl), 0.9 μl T4 gene 32 protein (gp32; 5μg/μl), and 3.1 μdH₂O, and was then added to the Component A followingthe addition of MutL. The exo⁻ Klenow Fragment is commercially available(New England Biolabs, Inc., (Beverly, Mass.)) and T4 gene 32 protein isalso commercially available (Roche Applied Science, (Indianapolis,Ind.)). The reaction continued for 30 minutes at 37° C. and was thenterminated by addition of 12.5 μl stop-buffer (1% SDS, 0.05 M EDTA, 30%glycerol, 0.2% Bromophenol blue). Reaction products were analyzed on a3% Genomic Performance Grade (GPG) low-melting-point (LMP) agarose gel(American Bioanalytical (Natick, Mass.)) in Tris Borate EDTA (TBE)buffer and ethidium bromide (FIG. 2B). A DNA fragment about of 120 bpwas observed (FIG. 2B), which matched the predicted product size of 123bp (FIG. 2A).

Example III Amplication of a Specific Sequence from Plasmid DNA by HAD

To test whether HDA can be used to amplify a specific target sequencefrom a DNA template, we used two pUC19/M13 universal primers,primer-1224 and primer-1233, to amplify a 110-bp sequence from a 2647-bpDNA plasmid, pAH1 (FIG. 15 (SEQ ID NO:9)) using the UvrD HDA system.Primer-1224 and primer-1233 are commercially available and theirsequence can be obtained at the company (New England Biolabs, Inc.,(Beverly, Mass.)). The amplification scheme is outlined in FIG. 3.

Two acetate-based reaction buffers were pre-made: 10×HDA Buffer Acontains 350 mM Tris-Acetate (pH7.5) and 100 mM DTT; 10×HDA Buffer Bcontains 10 mM Tris-Acetate (pH7.5), 1 mg/ml BSA, and 90 mM MagnesiumAcetate. The HDA reaction Component A was set up by combining:

5 μl 10×HDA Buffer A

1.5 μl of 23 nM AhdI-cleaved pAH1 plasmid

1 μl of 10 μM primer-1224

1 μl of 10 μM primer-1233

2 μl dNTPs (10 mM)

1.5 μl ATP (100 mM)

8 μl dH₂O

The reaction Component A was heated for 2 min at 95° C. to denature thetemplate, 3 min at 69° C. to anneal primers and 2 min at 37° C. beforeadding Component B.

Fifteen μl of reaction Component B was prepared by mixing:

5 μl 10×HDA Buffer B

1 μl exo⁻ Klenow Fragment (5 units/μl)

0.5 μl UvrD helicase (200 ng/μl)

1 μl MutL protein (400 ng/μl)

0.9 μl T4 gp32 (5 μg/μl)

21.6 μl dH₂O

Component B was then added to the Component A. The reaction wascontinued for one more hour at 37° C. and was then terminated byaddition of 12.5 μl stop-buffer (1% SDS, 0.05 M EDTA, 30% glycerol, 0.2%Bromophenol blue). Reaction products were analyzed on a 2% GPG LMP gelcontaining ethidium bromide (FIG. 4).

A 110-bp amplification product was observed on the 2% agarose gel. Thesize of this product matched the predicted length of the target sequence(FIG. 4, lane 1). In the absence of UvrD helicase, no amplification wasobserved confirming that helicase is required for the amplification.Moreover, the results indicated that UvrD was substantially moreeffective at amplifying target DNA in the presence of MutL and T4Gp32SSB.

Example IV Method of Amplification of Various Target Sequences from DNAPlasmids

To test whether the UvrD based HDA system was capable to amplify varioustarget sequences, several parallel reactions were carried out usingpAH1-derived plasmids containing different sequences and sizes ofinserts between primer 1224 and primer 1233.

A 50 μl HDA reaction was set up using two reaction Components, A and B,described below and mixing them in a sequential order. Two acetate-basedreaction buffers were pre-made: 10×HDA Buffer A contains 350 mMTris-Acetate (pH7.5) and 100 mM DTT; 10×HDA Buffer B contains 10 mMTris-Acetate (pH7.5), 1 mg/ml BSA, and 100 mM Magnesium Acetate.

Thirty five μl of Component A was made by combining:

5 μl 10×HDA Buffer A

1 μl pAH1 plasmid or pAH1 derivatives (50 ng/μl)

1 μl of 10 μM primer-1224

1 μl of 10 μM primer-1233

10 μl dNTPs (2 mM)

1.5 μl ATP (100 mM)

15.5 μl dH₂O

Fifteen μl of reaction Component B was prepared by mixing:

5 μl 10×HDA Buffer B

1 μl exo⁻ Klenow Fragment (5 units/μl)

0.5 μl UvrD helicase (200 ng/μl)

0.5 μl MutL (800 ng/μl)

0.9 μl T4 gp32 (5 μg/μl)

7.1 μl dH₂O

The HDA reaction was started by heating Component A at 95° C. for 2 minto denature the template. The Component A was then incubated for 3 minat 69° C. to anneal primers and 2 min at 37° C. to cool down thereaction. Fifteen μl of freshly made Component B was added to 35 μlComponent A following the denaturation, annealing, and cooling steps.The reaction continued for one more hour at 37° C. and was thenterminated upon addition of 12.5 μl stop-buffer (1% SDS, 0.05 M EDTA,30% glycerol, 0.2% Bromophenol blue). Amplification products werevisualized on a 3% GPG LMP agarose gel in TBE buffer and ethidiumbromide (FIG. 4). All of the amplification products matched thepredicted target sizes (FIG. 4, lanes 1-5). In addition, the UvrD-basedHDA system was able to amplify a target DNA as large as 650 bp in a HDAreaction (FIG. 4, lane 5).

Example V Amplication of a Specific Sequence from Bacterial Genomic DNAby HAD

HDA can also be used to amplify a specific target sequence from morecomplicated nucleic acid samples, such as viral genomic DNA or RNA,bacterial genomic DNA or human genomic DNA. In this example, we disclosea method to amplify and detect a specific target sequence from abacterial genome of an oral pathogen, Treponema denticola ATCC No.35405, using the E. coli UvrD-based HDA system. A restrictionendonuclease gene earIR was chosen as the target gene (FIG. 16 (SEQ IDNO:10)), and one 5′-primer and two 3′-primers were designed to hybridizeto the sequence of earIR gene. The reaction buffers and protocol weremodified for genomic DNA amplification. 10×HDA Buffer A contains 350 mMTris-Acetate (pH7.5) and 100 mM DTT. 10×HDA Buffer B contains 10 mMTris-Acetate (pH7.5), 1 mg/ml BSA, and 100 mM Magnesium Acetate.

The 20 μl Component A was set up by combining:

-   -   5 μl 10×HDA Buffer A    -   2 μl of Treponema denticola genomic DNA (50 ng/μl)    -   2 μl of 10 μM primer-58861 (5′ CCAAATGATGCTTATG TTGCTT 3′ (SEQ        ID NO:11))    -   2 μl of 10 μM primer-58862 (5′ CATAAGCCTCTCTTGGAT CT 3′ (SEQ ID        NO:12))    -   or 2 μl of 10 μM primer-58863 (5′ TCCACATCTTTCACAT TTCCAT 3′        (SEQ ID NO:13)    -   2 μl dNTPs (10 mM)    -   7 μl dH₂O

Thirty μl of reaction Component B was prepared by mixing:

5 μl 10×HDA Buffer B

4 μl 100 mM ATP

0.5 μl UvrD helicase (200 ng/μl)

0.5 μl MutL (800 ng/μl)

0.9 μl T4 gp32 (5 μg/μl)

1 μl exo⁻ Klenow Fragment (5 units/μl)

18.1 μl dH₂O

The reaction Component A was heated for 10 min at 95° C., 1 min at 53°C., and 2 min at 37° C. The freshly made Component B was then added toComponent A after it cooled down to 37° C. The reaction was continuedfor two more hours at 37° C. and was then terminated by addition of 12.5μl stop-buffer. Reaction products were analyzed on a 3% GPG LMP agarosegel (FIG. 5A). The predicted size of the target DNA is 97 bp betweenprimer-58861 and primer-58862 (FIG. 5A, lane 1), and the predictedlength between primer-58861 and primer-58863 is 129 bp (FIG. 5A, lane2). Two products were observed on an agarose gel and both matched thepredicted sizes of the target DNA. The amplification products weresequenced and the sequencing results confirmed that both match thesequence of the target DNA.

To test whether the UvrD helicase preparation can work with differentDNA polymerases, the HDA reaction was carried out to amplify the 129-bptarget sequence from T. denticola genome using UvrD helicase preparationand T7 Sequenase (USB, (Cleveland, Ohio)).

The reaction Component A (20 μl) was prepared by mixing:

-   -   5 μl 10×HDA Buffer A    -   2 μl of Treponema denticola genomic DNA (50 ng/μl)    -   2 μl of 10 μM primer-58861 (5′ CCAAATGATGCTTA TGTTGCTT 3′ (SEQ        ID NO:11))    -   2 μl of 10 μM primer-58863 (5′ TCCACATCTTTCACAT TTCCAT 3′ (SEQ        ID NO:13))    -   2 μl dNTPs (10 mM)    -   7 μl dH₂O

Thirty μl of reaction Component B was prepared by mixing:

5 μl 10×HDA Buffer B

4 μl 100 mM ATP

0.5 μl UvrD helicase (200 ng/μl)

0.5 μl MutL (800 ng/μl)

0.9 μl T4 gp32 (5 μg/μl)

1 μl T7 Sequenase (1.5 units/μl, or 3.5 units/μl)

18.1 μl dH₂O

The HDA reaction was carried out same as described above. Reactionproducts were analyzed on a 3% GPG LMP agarose gel (FIG. 5B). Anamplified product around 130 bp was observed on an agarose gel and itmatched the predicted sizes of the target sequence of 129 bp (FIG. 5B,lanes 1 and 2).

Example VI Amplifying Target Sequence from Human Genomic DNA Samples byHAD

In this example, we disclose a method to amplify a target sequence fromhuman genomic DNA sample using the E. coli UvrD-based HDA system. Humangenomic DNA prepared from a breast cancer cell line was purchased fromATCC No. 45537. Two primers, which are specific to the human DNAmethyltransferase gene (dnmt1), were synthesized. Different amounts ofinitial human genomic DNA were tested in the reaction, using genomic DNAat different concentrations: 50, 75, 100 ng/μl.

The 20 μl Component A was set up by combining:

-   -   5 μl 10×HDA Buffer A    -   2 μl of Human genomic DNA (50 to 100 ng/μl)    -   2 μl of 10 μM primer-dnmt5(′ 5′ GGAAGCTGCTAAGG ACTAGTT 3′ (SEQ        ID NO:14))    -   2 μl of 10 μM primer-dnmt3 (5′ CCATGTACCACAC ATGTGAAC 3′ (SEQ ID        NO:15))    -   2 μl dNTPs (10 mM)    -   7 μl dH₂O

Thirty μl of Component B was prepared by mixing:

5 μl 10×HDA Buffer B

3 μl 100 mM ATP

1 μl exo⁻ Klenow Fragment (5 units/μl)

0.5 μl UvrD helicase (200 ng/μl)

0.5 μl MutL (800 ng/μl)

0.9 μl T4 gp32 (5 μg/μl)

19.1 μl dH₂O

The reaction Component A was heated for 10 min at 95° C., 1 min at 53°C., and 2 min at 37° C. Component B was then added to the Component Aafter it cooled down to 37° C. The reaction was continued for two morehours at 37° C. and was then terminated by addition of 12.5 μlstop-buffer. Reaction products were analyzed on a 3% GPG LMP agarose gel(FIG. 6). A band of around 124 bp could be detected by ethidium bromidestaining and its size was in agreement with the length of the target inthe initial dnmt1 gene.

Example VII Amplification of a Target Sequence from an RNA Sample by HDA

In this Example, we disclose a method to amplify a target sequence fromRNA samples. Rat total RNA was used as nucleic acid substrate and wasfirst converted to a single stranded cDNA product using The ProtoScriptKit from New England Biolabs (Beverly, Mass.):

The reaction was set up by combining:

2 μl Rat total RNA (0.5 μg/μl)

2 μl primer dT₂₃VN (50 μM, New England Biolabs (Beverly, Mass.))

4 μl dNTP (2.5 mM)

8 μl H2O

and was incubated at 70° C. 5 min, then kept on ice.

After that, the following reagents were then added to the reaction tube:

2 μl 10×RT buffer (New England Biolabs (Beverly, Mass.))

1 μl RNase inhibitor (10 u/μl)

1 μl M-MulV reverse transcriptase (25 u/μl)

The RT reaction was incubated at 42° C. for 1 hr, followed by 95° C. for5 min. Two μl of the single-stranded cDNA product was added intoComponent A in HDA which was started by combining:

-   -   5 μl 10×HDA Buffer A    -   2 μl of first strand cDNA product    -   1 μl of 10 μM primer-sfo (5′ ACCGCATCGAATGCATG        TGGATCTCACCACCAACTGCTTAGC 3′ (SEQ ID NO:16))    -   1 μl of 10 μM primer-sre (5′ CGATTCCGCTCCAGACTTGGAT        CTGATGGCATGGACTGTGGT 3′ (SEQ ID NO:17))    -   2 μl dNTPs (10 mM)    -   9 μl dH₂O

Thirty μl of reaction Component B was prepared by mixing:

5 μl 10×HDA Buffer B

2 μl 100 mM ATP

1 μl exo⁻ Klenow Fragment (5 units/μl)

0.5 μl UvrD helicase (200 ng/μl)

0.5 μl MutL (800 ng/μl)

0.9 μl T4 gp32 (5 μg/μl)

20.1 μl dH₂O

The reaction Component A was heated for 2 min at 95° C., 1 min at 53°C., and 2 min at 37° C. Fresh-made Component B was then added to theComponent A after it cooled down to 37° C. The reaction was continuedfor two more hours at 37° C. and was then terminated by addition of 12.5μl stop-buffer. Amplification products were analyzed on a 3% GPG LMP gel(FIG. 7). A band of around 130 bp was observed in the agarose gel inagreement with the predicted size of 136 bp. The amplification productwas purified from the agarose gel and sequenced. The sequence ofamplification product matched the sequence of the initial target of therat glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) gene, confirmingthat the amplification was sequence specific.

Example VIII HDA can Amplify and Detect a Target Sequence from as Low as10 Copies of Bacterial Genomic DNA

To determine the amplification power of HDA, we performed HDA reactionswith various amounts of Treponema denticola genomic DNA. Each reactionwas carried out as detailed in Example V, except the amount of genomicDNA. The first tube contained 100 ng of Treponema denticola genomic DNAcorresponding to about 10⁷ copies of the Treponema denticola genome, and10-fold serial dilutions were carried out until 10 copies of Treponemadenticola genome were reached.

The UvrD-based HDA reactions were performed using primer-58861 andprimer-58862 in Example V. Reaction products were analyzed on a 3% GPGLMP agarose gel (FIG. 8). In general, the intensities of the 97-bp HDAproducts decrease as a function of decreasing initial copy number (FIG.8). A reaction performed without addition of target shows a faint bandin FIG. 8 probably due to contamination of reagents. It is extremelydifficult to maintain reagents free of target DNA contamination on ascale of 10 molecules. However, the intensity is still significantlyhigher than the background even at about 10 copies of initial target,suggesting that HDA is capable of amplifying single copy targetsequence. With 10 copies of initial target, about 10 ng products weregenerated by HDA, which corresponds to 10¹⁰ molecules of 97-bp fragment.Thus the HDA method disclosed here is capable of achieving over a onebillion-fold amplification.

Example IX Amplification of a Target Sequence from Bacterial Genomic DNAby HDA Without Heat Denaturation

Most of the isothermal target amplification methods start with a heatdenaturation step so that sequence-specific primers can anneal to thetarget sequence. Circumvention of the heat-denaturation step simplifiesthe amplification procedure. Accordingly, the UvrD-based HDA reactionwas carried out at 37° C. without the initial denaturation/annealingstep. The HDA reaction Component A was set up by combining the followingreagents in one tube:

-   -   5 μl 10×HDA Buffer A    -   2 μl of Treponema denticola genomic DNA (50 ng/μl)    -   2 μl of 10 μM primer-58861 (5′ CCAAATGATGCTTATG TTGCTT 3′ (SEQ        ID NO:11)    -   2 μl of 10 μM primer-58862 (5′ CATAAGCCTCTCTT GGATCT 3′ (SEQ ID        NO:12))    -   2 μl dNTPs (10 mM)    -   5 μl 10×HDA Buffer B    -   3 μl 100 mM ATP    -   0.9 μl exo⁻ Klenow Fragment (5 units/μl)    -   0.5 μl UvrD helicase (200 ng/μl)    -   0.5 μl MutL (800 ng/μl)    -   0.9 μl T4 gp32 (5 μg/μl)    -   26.2 μl dH₂O

and the 50 μl reaction was then incubated for two hours at 37° C. Thereaction was then terminated by addition of 12.5 μl stop-buffer.Amplification products were analyzed on a 3% GPG LMP agarose gel (FIG.9). The size of the amplification product matches the predicted size ofthe target DNA (97 bp).

Example X Method of Amplification of Long Target Sequences by HDA UsingReplicative Helicase (T7 Gene 4 Helicase)

To test whether a hexameric replicative helicase, such as T7 Gp4Bhelicase, can be used to amplify a longer target sequence and to testwhether different HDA systems can be used to perform HDA reaction, theT7 Gp4B helicase preparation was used along with the T7 Sequenase (USB,(Cleveland, Ohio)) to amplify a 2.3-kb target sequence. This targetsequence was the E. coli Rep gene (GenBank Accession No. U00096), whichwas cloned into plasmid pCR2.1 (Invitrogen Corporation) and theresulting recombinant plasmid was named pCR-Rep. Primer 1224 and primer1233, flanking the insertion site, were used to amplify the 2.3-kbtarget. A 50 μl HDA reaction was set up using two reaction Components, Aand B, described below and mixing them in a sequential order. Twoacetate-based reaction buffers were pre-made: 10×HDA Buffer A contains350 mM Tris-Acetate (pH7.5) and 100 mM DTT; 10×HDA Buffer B contains 10mM Tris-Acetate (pH7.5), 1 mg/ml BSA, and 100 mM Magnesium Acetate.Three parallel tubes were set up each contained 20 μl of reactionComponent A by mixing the following Components in each tube:

5 μl 10×HDA Buffer A

1 μl of plasmid pCR-Rep (50 ng/μl)

1 μl of 10 μM primer-1224

1 μl of 10 μM primer-1233

3 μl dNTPs (10 mM)

9 μl dH₂O

Three parallel tubes were prepared each contained 30 μl of reactionComponent B by mixing the following Components in each tube:

5 μl 10×HDA Buffer B

9.3 μl Helicase preparation*

1 μl T7 Sequenase (1 u/μl, USB Corporation)

14.7 μl dH₂O

*Three different helicase preparations were used in HDA reactions. Thefirst one was a T7 helicase preparation which contained 4.5 μl T7 Gp4Bhelicase (70 ng/μl), 1.3 μl T7 Gp2.5 SSB (5 μg/μl), 1.5 μl of 100 mMdTTP, and 2 μl H₂O (FIG. 5, lane 1). The second helicase preparationcomprised a plurality of two helicases and it contained 4.5 μl T7 Gp4Bhelicase (70 ng/μl), 0.5 μl E. coli UvrD helicase (200 ng/μl), 0.5 μlMutL (800 ng/μl), 1.3 μl T7 Gp2.5 SSB (5 μg/μl), 1.5 μl of 100 mM dTTP,and 1 μl of 100 mM ATP (FIG. 5, lane 2). The third one was a negativecontrol which contained 1.3 μl T7 Gp2.5 SSB (5 μg/μl), 1.5 μl of 100 mMdTTP and 6.5 μl H₂O (FIG. 5, lane 3).

HDA reactions were started by heating three tubes, each containingidentical 20-μl Component A, at 95° C. for 2 min to denature thetemplate and then at 37° C. for 1 min to hybridize the primers. Threefreshly made Component B mixtures, each containing a different helicasepreparation, were then added to each of the Component A mixtures. Thereaction continued for two more hours at 37° C. and was then terminatedupon addition of 12.5 μl stop-buffer (1% SDS, 0.05 M EDTA, 30% glycerol,0.2% Bromophenol blue). Amplification products were visualized on a 1%agarose gel in TBE buffer and ethidium bromide (FIG. 10). In thepresence of T7 Gp4B helicase preparation, an amplification productaround 2.3 kb was observed and it matched the predicted target size(FIG. 10, lane 1). In the presence of a helicase preparation comprisedof T7 Gp4B helicase and E. coli UvrD helicase, a similar 2.3-kb productwas observed (FIG. 10, lane 2). In addition, no amplification productwas observed in the negative control, in which no helicase was presentin the helicase preparation (FIG. 10, lane 3). The amplificationproducts from lane 1 and lane 2 were later sequenced and the sequencingresults confirmed that the products were derived from the Rep gene.

Example XI Method of Amplification of DNA Fragment by HDA Using RecBCD

A nuclease-deficient mutant RecB^(D1067A)CD (Wang et al., J. Biol. Chem.275:507-513 (2000)) was used in a HDA reaction to amplify a 400-bp DNAfragment. This blunt-end dsDNA template was generated by a PCR reactionusing a pUC19-derivative containing a 400-bp insert between primer-1224and primer-1233 (New England Biolabs, Inc. (Beverly, Mass.)). Thecloning and purification of the RecB^(1067A)CD protein has beendescribed previously (Wang et al., J. Biol. Chem. 275:507-513 (2000)). A50-μl reaction was set up by combining the following reagents in onetube:

-   -   5 μl 10×HDA Buffer (360 mM Tris-Acetate (pH7.5), 250 mM KOAC,        100 mM DTT, 1 mg/ml BSA, and 50 mM Magnesium Acetate)    -   1 μl of 400-bp template (2 ng/μl)    -   1.5 μl of 10 μM primer-1224    -   1.5 μl of 10 μM primer-1233    -   2 μl dNTPs (10 mM)    -   2 μl 100 mM ATP    -   1 μl Sequenase Version 2.0 (1.3 unit/μl)    -   0.5 μl RecB^(D1067A)CD helicase (130 ng/μl)    -   1.3 μl T4 gp32 (3.8 μg/μl)    -   26.2 μl dH₂O

The 50 μl reaction was incubated for one hour at 37° C. and thenterminated by addition of 12.5 μl stop-buffer. Amplification productswere analyzed on a 1% agarose gel (FIG. 11, lane 2). The size of theamplification product matches the predicted size of the target DNA (400bp). A control reaction without RecB^(D1067A)CD helicase did not give aproduct (FIG. 11, lane 3).

Example XII Method of Thermostable Helicase-Dependent Amplification of aSpecific Sequence

Performing HDA at high temperature using a thermostable helicase and athermostable polymerase may increase the specificity of primer bindingand improve the specificity of amplification. In this example, wedisclose a method to amplify and detect a specific target sequence froma bacterial genome of an oral pathogen, Treponema denticola ATCC No.35405, using the Tte-UvrD-based thermostable Helicase-DependentAmplification or t-HDA system.

1. Obtaining a Thermostable Helicase

A thermostable UvrD-like helicase, Tte-UvrD, was cloned and purifiedfrom a completely sequenced thermostable bacterium, Thermoanaerobactertengcongensis (Bao, et al., Genome Res. 12:689-700 (2000)). Thenucleotide sequence of the UvrD gene of T. tengcongensis, which encodesthe Tte-UvrD helicase, is located between positions 605,527 and 607,668of the T. tengcongensis genome and the sequence can be found in GenBank(Accession No.: NC_(—)003869; Bao, et al., Genome Res. 12:689-700(2000)). PCR was used to amplify the Tte-UvrD gene using T.tengcongensis genomic DNA (100 ng) plus primer TUF(5′-ATACATATGATTGGAGTGAAAAAGATGAA-3′ (SEQ ID NO:18)) and primer TUR(5′-AAATAAGCTCTTCAG CAAGAAATTGCCTTAATAGGAG-3′ (SEQ ID NO:19)). Theprimers contained restrictions enzymes sites that allowing the cloningof the Tte-UvrD gene into the NdeI and SapI sites of pTYB1 (New EnglandBiolabs, Inc., (Beverly, Mass.)). PCR products was digested with NdeIand SapI and then was ligated to the digested pTYBI. Ligation productswere transformed into ER2502 cells. Positive transformants were screenedby selective growth on LB plates containing 100 μg/ml ampicillin,followed by colony PCR and sequencing of the insert. After analysis ofsequencing results, correct constructs were transformed into E. coliER2566 cells. ER2566 cells containing pTYB1-Tte-UvrD were grown at 37°C. in LB media supplemented with 100 ug/ml ampicillin. When OD₅₅₀reached ˜0.65, protein expression was induced with 0.4 mM IPTG. After anovernight incubation at 15° C., cells were harvested by centrifugation.

The chitin binding domain (CBD) of the intein tag allowed affinitypurification of the fusion proteins on a chitin bead column (New EnglandBiolabs, Inc., (Beverly, Mass.)). The Tte-UvrD helicase was firstpurified using a chitin column and the protocol is described in detailin Example I (UvrD and MutL purification). Next, Tte-UvrD was furtherpurified by a 1-ml heparin TSK column (Pharmacia (Piscataway, N.J.)).Fractions containing Tte-UvrD were analyzed by SDS-PAGE. The purefractions were pooled and dialyzed overnight against storage buffer (20mM Tris-HCl (pH8.2), 200 mM NaCl, 1 mM EDTA, 1 mM EGTA, 15 mM2-mercaptoethanol, 50% glycerol). The final concentration was determinedusing the Bradford protein assay (Bradford Anal. Biochem. 72:248-254(1976)) and SDS-PAGE.

2. Thermostable Helicase-Dependent Amplification (t-HDA)

The purified thermostable Tte-UvrD helicase was used with a thermostableBst DNA polymerase large fragment (New England Biolabs, Inc., (Beverly,Mass.)) to selectively amplify a target sequence from genomic DNA athigh temperature. A restriction endonuclease gene earIR was chosen asthe target gene (FIG. 16 (SEQ ID NO:10)). Two primers, one for each endof the target fragment, were designed and they have high meltingtemperature (˜75° C.) so that they can hybridize to the target sequenceat high temperature. The reaction buffers and protocol were modified forgenomic DNA amplification. Reaction buffer is 10× ThermoPol reactionBuffer (New England Biolabs, Inc., (Beverly, Mass.)): 200 mM Tris-HCl(pH8.8), 100 mM KCl, 100 mM (NH₄)₂SO₄, 20 mM MgSO₄, 1% Triton X-100.

Thirty-five μl of Component A was made by combining:

-   -   3.5 μl 10× ThermoPol Buffer    -   2 μl of 0.83 μM Treponema denticola genomic DNA    -   1 μl of 10 μM primer p5-76 (5′-GGCCAGTTTGAA TAAGACAATGAATTATT-3′        (SEQ ID NO:20))    -   1 μl of 10 μM primer p3-76 (5′-ATTTTGAAACACA        AGAATGGAAATGTGAAAG-3′ (SEQ ID NO:21))    -   2 μl dNTPs (10 mM)    -   1.5 μl dATP (100 mM)    -   24 μl dH₂O

Fifteen μl of reaction Component B was prepared by mixing:

1.5 μl 10× ThermoPol Buffer

2.6 μl Bst DNA Polymerase, Large Fragment (8 units/μl)

1 μl UvrD-tte helicase (100 ng/μl)

0.9 μl T4 gp32 (5 μg/μl)

9 μl dH₂O

The HDA reaction was started by heating Component A at 95° C. for 2 minto denature the template. The Component A was then cooled down to 60°C., kept at 60° C. for 3 min to anneal primers. Fifteen μl of freshlymade Component B were added to 35 μl Component A following thedenaturation and annealing steps. The reaction continued for one morehour at 60° C. and was then terminated upon addition of 12.5 μlstop-buffer (1% SDS, 0.05 M EDTA, 30% glycerol, 0.2% Bromophenol blue).Amplification products were visualized on a 2% GPG LMP agarose gel inTBE buffer and ethidium bromide. The size of the amplified DNA matchesthe predicated target size of 82 bp (FIG. 12).

Example XIII Method of Amplification and Detection of a SpecificSequence from Neisseria Gonorrhoeae by t-Had

In this Example, we disclose a method to amplify and detect a specifictarget sequence from a different bacterial genome, Neisseriagonorrhoeae. N. gonorrhoeae is a human pathogen which causes gonorrhea,one of the most common sexually transmitted diseases. N. gonorrhoeaegenomic DNA was purchased from American Type Culture Collection (ATCCNo. 700825, (Manassas, Va.)). Two primers, one for each end of thetarget sequence (CATATGTAACAGCAGGTCAGGCCATATCCAATATTCCACAAAATGCCAGTAATAATGAATTACTGAAAATCAGCGATA AAACACGCCGTATGTTG (SEQID NO:22)), were synthesized and they have a melting temperature of ˜78°C. The reaction buffers and protocol were modified for genomic DNAamplification. Reaction buffer is 10× ThermoPol reaction Buffer (NewEngland Biolabs, Inc., (Beverly, Mass.)).

Thirty-five μl of Component A was made by combining:

-   -   3.5 μl 10× ThermoPol Buffer    -   2 μl of N. gonorrhoeae genomic DNA (50 ng/μl)    -   1 μl of 10 μM primer H153 (5′-CATATGTAACAGCAGGT CAGGCCATAT-3′        (SEQ ID NO:23)    -   1 μl of 10 μM primer H154 (5′-CAACATACGGCGT GTTTTATCGCTGAT-3′        (SEQ ID NO:24)    -   2 μl dNTPs (10 mM)    -   1.5 μl dATP (100 mM)    -   24 μl dH₂O

Fifteen μl of reaction Component B was prepared by mixing:

1.5 μl 10× ThermoPol Buffer

2.6 μl Bst DNA Polymerase, Large Fragment (8 units/μl)

1 μl UvrD-tte helicase (100 ng/μl)

0.9 μl T4 gp32 (5 μg/μl)

9 μl dH₂O

The HDA reaction was started by heating Component A at 95° C. for 2 minto denature the template. The Component A was then cooled down to 60°C., kept at 60° C. for 3 min to anneal primers. Fifteen μl of freshlymade Component B was added to 35 μl Component A following thedenaturation and annealing steps. The reaction continued for one morehour at 60° C. and was then terminated upon addition of 12.5 μlstop-buffer (1% SDS, 0.05 M EDTA, 30% glycerol, 0.2% Bromophenol blue).Amplification products were visualized on a 2% GPG LMP agarose gel inTBE buffer and ethidium bromide. In the presence of the Tte-UvrDhelicase, Gp32 SSB, and the large fragment of Bst DNA polymerase, adominant band around 95-bp was observed on the gel and it matches thepredicated target size (FIG. 13, lane 1). Gp32 SSB was eliminated in aparallel reaction and the 95-bp product was also observed (FIG. 13, lane2), suggesting that single-stranded DNA binding protein is not requiredfor this HDA system. When the Tte-UvrD helicase is absent from thereaction, no amplification is observed (FIG. 13, lane 3), which furtherconfirms that this is a helicase-dependent amplification. This exampledemonstrates that HDA requires a minimal of two enzymatic activities, aDNA helicase activity and a DNA polymerase activity.

Example XIV Real Time Detection of a Target Sequence of PathogenicBacteria in a Sample

HDA can be combined with other technologies and can be used for genometyping such as determining single nucleotide polymorphisms (SNP) and forthe identification of infectious agents. For example, HDA can be coupledwith other nucleic acid detection methods, such as fluorescent-labeledLUX™ Primers (Invitrogen Corporation, Carlsbad, Calif.) and a real-timefluorescent detection system (iCycler, Bio-Rad Laboratories Inc.,Hercules, Calif.), to amplify and detect the presence of a targetsequence in real time. This example illustrates real-time amplificationand detection of a target sequence (FIG. 16 (SEQ ID NO:10)) in abacterial pathogen, Treponema denticola (ATCC No. 35405), using HDAmethod and the UvrD HDA system. The fluorescent-labeled primer,primer-175-LUX (5′ cacatttTGAAACACAAGAATGGAAATGTG 3′ (SEQ ID NO:25)),was customer designed based on the target sequence (FIG. 16 (SEQ IDNO:10)) and obtained from Invitrogen Corporation. The reaction bufferswere pre-made: 10×HDA Buffer A contains 350 mM Tris-Acetate (pH7.5) and100 mM DTT. 10×HDA Buffer B contains 10 mM Tris-Acetate (pH7.5), 1 mg/mlBSA, and 100 mM Magnesium Acetate.

To test the reproducibility of real-time HDA reaction, two parallelreactions were carried out (FIG. 12, line 1 and line 2). Each reactionwas set up as following: 20 μl Component A was made by combining:

-   -   5 μl 10×HDA Buffer A    -   1 μl of Treponema denticola genomic DNA (30 ng/μl)    -   2 μl of 10 μM primer-175-LUX (5′ cacatttTGAAACACAAG AATGGAAATGTG        3′ (SEQ ID NO:25))    -   2 μl of 10 μM primer-175-Rev (5′ GGCCAGTTTGAAT AAGACAATG 3′ (SEQ        ID NO:26))    -   2 μl of 10 mM dNTPs    -   8 μl dH₂O

Thirty μl of reaction Component B was prepared by mixing:

5 μl 10×HDA Buffer B

1.5 μl 100 mM ATP

1 μl exo⁻ Klenow Fragment (5 units/μl)

0.5 μl UvrD helicase (200 ng/μl)

0.8 μl MutL (800 ng/μl)

1.2 μl T4 gp32 (5 μg/μl)

20 μl dH₂O

The reaction Component A was incubated for 2 min at 95° C. and then 1min at 37° C. The freshly made Component B was then added to Component Aafter it cooled down to 37° C. The reaction was continued in an iCycler(Bio-Rad) at 37° C. The amplification product was detected in real-timeby measuring fluorescent signals (490 nM for FAM) at a 5-min intervalusing a real-time PCR machine, iCycler (Bio-Rad). Fluorescent signalsfrom reactions 1 and 2 started to increase at 40 minutes and crossedT_(t) (time of threshold) line around 50 minutes (FIG. 14, lines 1 and2). The T_(t) value for these two reactions were about 50 minutes. Inaddition, the curves derived from reaction 1 and reaction 2 were verysimilar, suggesting the reproducibility of real-time HDA reaction wasgood. In the negative control, fluorescent signal remained below T_(t)line (FIG. 14, line 3).

Example XV Two-Reaction Vessel/Two-Buffer RT-HDA

In this example, human total RNA was used as nucleic acid substrate andwas first converted to a single stranded cDNA product using theProtoScript® Kit from New England Biolabs (New England Biolabs, Inc.,Ipswich, Mass.).

The reaction was set up by combining:

1 μl human total RNA (1 μg/μl)

2 μl primer dT23VN (50 μM, New England Biolabs, Inc., Ipswich, Mass.)

4 μl dNTP (2.5 mM)

9 μl nuclease-free H₂O

and started by incubated at 70° C. for 5 min, then kept on ice.

The following reagents were then added to the reaction tube:

2 μl 10×RT buffer (New England Biolabs, Inc., Ipswich, Mass.)

-   -   1 μl RNase inhibitor (10 units/μl)    -   1 μl M-MuLV reverse transcriptase (25 units/μl)

The RT reaction was incubated at 42° C. for 1 hr, followed by 95° C. for5 min. 1 μl of RNase H (2 units/μl) was then added and the RT reactionwas further incubated at 37° C. for 20 min, followed by 95° C. for 5min. The RT product was then diluted into 50 μl with dH2O. A serialdilution of this diluted RT product was then prepared. In each time, 5μl of the previously diluted RT product was further diluted into 50 μlwith dH2O. In the subsequent tHDA reaction, a pair of primers, GAPDF1and GAPDR1, specifically targeting the GAPDH gene was used foramplification. The tHDA reactions were started by combining thefollowing components in reaction mixture A with a total volume of 25 μl:

2.5 μl 10× Mg-free tHDA buffer (see below*)

5 μl of serial diluted RT product (0.0001% to 1% of RT product)

0.5 μl 10 μM Primer GAPDF1 (SEQ ID NO:27)

0.5 μl 10 μM Primer GAPDR1 (SEQ ID NO:28)

16.5 μl dH₂O

25 μl of reaction mixture B was also prepared by mixing:

2.5 μl 10× Mg-free tHDA buffer (see below*)

1.75 μl 100 mM MgSO4

2 μl 500 mM NaCl

2 μl 10 mM dNTP

1.5 μl 100 mM dATP

2.5 μl Bst DNA Polymerase, LF (8 units/μl; New England Biolabs, Inc.,Ipswich, Mass.)

1 μl Tte-UvrD helicase (100 ng/μl)

11.75 μl dH₂O

*10× Mg-free tHDA buffer contains 100 mM KCl, 100 mM (NH4)2SO4, 200 mMTris-HCl (pH 8.8 at 25° C.), 1% Triton X-100.

The reaction mixture A was heated for 2 min at 95° C., and then 3 min at65° C. Fresh-made mixture B was then added to the mixture A andincubated at 65° C. for 75 min. Amplification products were analyzed ona 2% agarose gel (FIG. 18). A band of around 100 bp was observed in lane2 (1% or 10 ng initial total RNA input) and lane 3 (0.1% or 1 ng initialtotal RNA input) on the agarose gel in agreement with the predicted sizeof 96 bp.

Example XVI One-Reaction Vessel/Two-Buffer RT-HDA

In this example, the first-strand cDNA synthesis was coupled with tHDAreaction in one tube using two-step incubation. Different amount ofhuman total RNA ranging from 10 pg to 100 ng and a pair of specificprimers, GAPDF1 and GAPDR1, of the GAPDH gene were used foramplification.

The reaction with a total volume of 50 μl was set up in one tube bycombining:

5 μl 10× Mg-free tHDA buffer

1 μl variable amount of human total RNA (10 pg-100 ng)

0.5 μl 10 μM Primer GAPDF1 (SEQ ID NO:27)

0.5 μl 10 μM Primer GAPDR1 (SEQ ID NO:28)

1.75 μl 100 mM MgSO4

2 μl 500 mM NaCl

2 μl 10 mM dNTP

1.5 μl 100 mM dATP

1 μl RNase inhibitor (10 units/μl)

1 μl M-MulV reverse transcriptase (25 units/μl)

2.5 μl Bst DNA polymerase, LF (8 units/μl, New England Biolabs, Inc.,Ipswich, Mass.)

1 μl Tte-UvrD helicase (100 ng/μl)

30.25 μl nuclease-free H2O

The RT-HDA reaction was carried out at 42° C. for 5 min, followed by 65°C. for 60 min. Amplification products were analyzed on a 2% agarose gel(FIG. 19). A strong band of around 100 bp was observed in the presenceof 100 ng initial total RNA (lane 3) and 10 ng of initial total RNA(lane 4) on the agarose gel. A weak band of around 100 bp was alsoobserved in the presence of 1 ng (lane 5) of initial total RNA on theagarose gel. The observed DNA fragment size was in agreement with thepredicted target size of 96 bp. When the amount of input total RNA wasbelow 1 ng, only none-specific amplification in the form of primer-dimerwas observed (lanes 6 to 8). In addition, when reverse transcriptase(lane 1) or helicase (lane 2) was omitted from the reaction, noamplification was observed, suggesting the amplification depended onboth RT and HDA functions.

Example XVII One-Reaction Vessel/One-Buffer RT-HDA

In this example, a reverse transcriptase with higher thermo-stabilitywas selected and the first-strand cDNA synthesis and thehelicase-dependent isothermal DNA amplification reaction were coupledand carried out in one tube and in one step. Different amount of humantotal RNA ranging from 100 pg to 100 ng and a pair of specific primers,GAPDF1 and GAPDR1, of the GAPDH gene were used for amplification.

The reaction with a total volume of 50 μl was set up in one tube bycombining:

5 μl Mg-free tHDA buffer

1 μl variable amount of human total RNA (100 pg-100 ng)

0.5 μl 10 μM Primer GAPDF1 (SEQ ID NO:27)

0.5 μl 10 μM Primer GAPDR1 (SEQ ID NO:28)

1.75 μl 100 mM MgSO4

2 μl 500 mM NaCl

2 μl 10 mM dNTP

1.5 μl 100 mM dATP

1 μl SuperScript™ III reverse transcriptase (200 units/μl, Invitrogen,Carlsbad, Calif.)

2.5 μl Bst DNA polymerase, LF (8 units/μl, New England Biolabs, Inc.,Ipswich, Mass.)

1 μl Tte-UvrD helicase (100 ng/μl)

31.25 μl nuclease-free H2O

The RT-HDA reaction was carried out in one step at 62.5° C. for 60 min.Amplification products were analyzed on a 2% agarose gel (FIG. 20). Inthe presence of 100 ng (lane 2) or 10 ng (lane 3) of initial total RNA,a DNA fragment of around 100 bp was observed on the agarose gel inagreement with the predicted target size of 96 bp. When the amount ofinput total RNA was below 10 ng, only none-specific amplification in theform of primer-dimer was observed (lanes 4-6).

Example XVIII Modified One-Buffer RT-HDA

In this example, a separate denaturation step involved in thedenaturation of RNA template and primers was applied to decreasenon-specific amplification. A reverse transcriptase with higherthermostability at 65° C. was selected and the first-strand cDNAsynthesis and the helicase-dependent isothermal DNA amplificationreaction were coupled and carried out in one tube and in one step.Different amount of human total RNA ranging from 2 pg to 200 ng and apair of specific primers, GAPDF3 and GAPDR3, located on different exonsof the GAPDH gene were used for amplification.

The reaction with a total volume of 50 μl was set up by first making MixA and Mix B. Mix A was prepared in one sterile microtube by combining:

2.5 μl 10×RT-HDA buffer (see below*)

2 μl variable amount of human total RNA (2 pg-200 ng)

0.375 μl 10 μM primer GAPDF3 (SEQ ID NO:29)

0.375 μl 10 μM primer GAPDR3 (SEQ ID NO:30)

19.75 μl nuclease-free H2O

Total volume: 25 μl

For the negative control reaction, replace 2 μl nuclease-free H2O withthe total RNA.

Mix B was prepared in another sterile microtube by combining:

2.5 μl 10×RT-HDA buffer (see below*)

1.75 μl 100 mM MgSO4

4 μl 500 mM NaCl

2 μl 10 mM dNTP

1.5 μl 100 mM dATP

0.5 μl RNase inhibitor (40 units/μl, New England Biolabs, Inc., Ipswich,Mass.)

0.7 μl ThermoScript reverse transcriptase (15 units/μl, Invitrogen)

2.5 μl Bst DNA Polymerase, LF (8 units/μl, New England Biolabs, Inc.,Ipswich, Mass.)

1 μl Tte-UvrD helicase (150 ng/μl)

8.55 μl nuclease-free H2O

Total volume: 25 μl

*10×RT-HDA buffer contains 100 mM KCl, 200 mM Tris-HCl (pH 8.8 at 25°C.).

Denaturation of Mix A was carried out at 95° C. for 2 min. After coolingMix A on ice, Mix B was added into Mix A. The RT-HDA reaction wascarried out in one step at 65° C. for 120 min. Amplification productswere analyzed on a 2% agarose gel (FIG. 21). In the presence of as highas 200 ng down to as low as 2 pg of initial total RNA (lanes 2-7), asingle DNA fragment of around 100 bp was observed on the agarose gel inagreement with the predicted target size of 95 bp. No none-specificamplification was observed from 2 ng to 200 ng of initial total RNA(lanes 2-7) or from the no template control (lane 8). This indicatesthat the modified one-step RT-HDA for detection of the GAPDH gene workswell in a range of at least 5 log of total RNA.

Example XIX Detection of Enterovirus RNA

In this example, the modified one-step RT-HDA method described inExample 4 was applied in detection of a RNA virus Enterovirus.Enterovirus RNAs purified from the Enterovirus Clear QC Panel (Argene,Varilhes, France) ranging from 40 copies to 4000 copies were used astemplates and a pair of specific primers, EVF1A and EVR1, targeting the5′-UTR sequence conserved in all Enterovirus species were used asprimers for amplification.

Purification of Enterovirus Clear QC Panel was carried out with a QIAampViral RNA Mini Kit (Qiagen, Valencia, Calif.). 140 μl of eachEnterovirus standard sample with a concentration of 10 copies/ml, 100copies/ml and 1000 copies/ml was used for purification and eluted into70 μl of elution buffer. The modified one-step RT-HDA reaction with atotal volume of 50 μl was set up by first making Mix A and Mix B. Mix Awas prepared in one sterile microtube by combining:

2.5 μl 10×RT-HDA buffer (see below*)

2 μl variable amount of Enterovirus RNA (40-4000 copies)

0.375 μl 10 μM primer EVF1A (SEQ ID NO:31)

0.375 μl 10 μM primer EVR1 (SEQ ID NO:32)

19.75 μl nuclease-free H2O

Total volume: 25 μl

For the negative control reaction, replace 2 μl nuclease-free H2O withthe Enterovirus RNA.

Mix B was prepared in another sterile microtube by combining:

2.5 μl 10×RT-HDA buffer (see below*)

1.75 μl 100 mM MgSO4

2 μl 500 mM NaCl

2 μl 10 mM dNTP

1.5 μl 100 mM dATP

0.5 μl RNase inhibitor (40 units/μl, New England Biolabs, Inc., Ipswich,Mass.)

0.7 μl ThermoScript reverse transcriptase (15 units/μl, Invitrogen)

2.5 μl Bst DNA Polymerase, LF (8 units/μl, New England Biolabs, Inc.,Ipswich, Mass.

1 μl Tte-UvrD helicase (150 ng/μl)

10.55 μl nuclease-free H2O

Total volume: 25 μl

*10×RT-HDA buffer contains 100 mM KCl, 200 mM Tris-HCl (pH 8.8 at 25°C.).

Denaturation of Mix A was carried out at 95° C. for 2 min. After coolingMix A on ice, Mix B was added into Mix A. The RT-HDA reaction wascarried out in one step at 65° C. for 120 min. Amplification productswere analyzed on a 2% agarose gel (FIG. 22). In the presence of 4000copies (lane 1) and 400 copies (lane 2) of initial Enterovirus RNA, asingle DNA fragment of around 120 bp was observed on the agarose gel inagreement with the predicted target size of 116 bp. No amplification wasobserved from 40 copies of initial Enterovirus RNA (lane 3).Non-specific amplification was not observed from 4000 copies to 40copies of initial Enterovirus RNA (lanes 1-3) or from the no templatecontrol (lane 4).

1. A method for helicase-dependent amplification of an RNA, comprising:(a) adding to the RNA, a mixture comprising a reverse transcriptase, ahelicase and a single strand binding protein unless the helicase is athermostable helicase in which case the single strand binding protein isnot required, a plurality of oligonucleotide primers and one or morepolymerases; and (b) in the mixture, reverse transcribing the RNA toform a cDNA and amplifying, by helicase-dependent amplification, thecDNA wherein the amplification does not occur in the absence of thehelicase as determined by gel electrophoresis.
 2. A method according toclaim 1, wherein the helicase preparation comprises a UVrD helicase orhomolog thereof.
 3. A method according to claim 2, wherein the UVrDhelicase is a thermostable helicase.
 4. A method according to claim 1,wherein the helicase-dependent amplification is isothermal.
 5. A methodaccording to claim 4, wherein the isothermal amplification occurs at atemperature in the range of 20° C.-75° C.
 6. A method according to claim1, wherein the polymerase is Bst polymerase.
 7. A method according toclaim 1, wherein the mixture further comprises an accessory protein.